Clavulanic acid sulphates

ABSTRACT

Clavulanic acid sulphates of the formula ##STR1## wherein M 1  is trimethylamine, dimethylalanine, pyridine, N-methylpyridine or N-methylmorpholino, are useful for their antibacterial activity and are also useful as intermediates for the production of antibacterially active compounds.

The present invention relates to novel β-lactam containing compounds which possess antibacterial activity and some of which are useful intermediates in the manufacture of antibacterially active compounds, to a process for the preparation of these novel compounds and to pharmaceutical compositions containing them.

Clarrilanic acid which is the compound of the formula (I): ##STR2## is produced by the aerobic cultivation of Streptomyces clavuligeris in conventional nutrient media at about 25° - 30° C under roughly neutral conditions and possess a β-lactamase inhibitory and antibacterial activity.

Clavulanic acid has the following properties:

A. It is a carboxylic acid.

B. It forms a sodium salt which has a characteristic infra-red spectrum substantially as shown in FIG. 1.

c. It is able to inhibit the growth of strains of Staphylococcus aureus.

D. It is able to synergyze the antibacterial effect of ampicillin against β-lactamase producing strains of Escherichia coli, Klebsiella aerognes and Staphylococcus aureus.

E. It is able to synergyze the antibacterial effect of cephaloridine against the β-lactamase producing strains of Proteus mirabilis and Staphylococcus aureus.

F. It forms a methyl ester which has a molecular weight (by mass spectroscopy) of 213.0635 which corresponds to the formula C₉ H₁₁ NO₅.

Thus clavulanic acid may be regarded as a monobasic carboxylic acid of the formula C₈ H₉ NO₅ which in the form of its sodium salt has a characteristic infra-red absorption spectrum substantially as shown in FIG. 1.

The compound produced by Streptomyces clavuligerus which has the above properties has the formula (II'): ##STR3##

Thus clavulanic acid may be named 3-(β-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0] heptane-2-carboxylic acid.

The stereochemistry at C₅ and C₂ of the clavulanic acid is the same as that found in naturally occurring penicillins and cephalosporins so that clavulanic acid may be represented by the structural formula (I'): ##STR4##

Thus a fuller chemical name for clavulanic acid is Z-(2R,5R)-3-(β-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3,2,0]heptane-2-carboxylic acid.

The great usefulness of clavulanic acid may be readily appreciated when it is realized that certain strains of Klebsiella aerogenes A, the growth of which is not inhibited by the presence of 125 μg/ml. of ampicillin, amoxycillin, carbenicillin or benzyl penicillin or by the presence of 10μg/ml. of clavulanic acid, are inhibited by the presence of less than 12.5 μg/ml. of the previously mentioned penicillins when 5 μg/ml. of clavulanic acid is also present. Similar results have been observed for combinations containing various esters of clavulanic acid. For example, strains of Klebsiella aerogenes A, the growth of which is not inhibited by 125 μg/ml. of ampicillin, or by 10 μg/ml of clavulanic acid methyl ester are inhibited by less than 12.5 μg/ml. of ampicillin in the presence of 5 μg/ml. of the clavulanic acid methyl ester. It has also been found that strains of Staphylococcus aureus Russell, the growth of which is not inhibited by the presence of 100 μg/ml. of ampicillin or by 5 μg/ml of clavulanic acid, are inhibited by the presence of less than 10 μg/ml. of ampicillin in the presence of 1 μg/ml. of clavulanic acid. In tests on female mice, it has been found that blood and tissue levels of clavulanic acid considerably in excess of 5 μg/ml. can readily be achieved by subcutaneous administration of 100 mg/kg of the sodium salt of clavulanic acid and that useful levels of clavulanic acid can be obtained after oral administration of 100 mg/kg of the sodium salt of clavulanic acid.

Most suitably, the salts of clavulanic acid will be pharmaceutically acceptable salts such as the sodium, potassium, calcium, magnesium, aluminium, ammonium and substituted ammonium salts such as the trimethylammonium, benzathine, procain and like salts conventionally formed with penicillins or cephalosporins. Non-pharmaceutically acceptable salts of clavulanic acid are also included as they are useful intermediates in the preparation of esters of clavulanic acid, for example, the lithium or silver salts of clavulanic acid may be reacted with benzyl bromide to form the useful benzyl ester of clavulanic acid.

Salts of clavulanic acid tend to be more stable than the parent acid per se and thus form a favoured aspect of this invention. Particularly suitable salts of clavulanic acid include the sodium and potassium salts which have the formula (III') and (IV') respectively: ##STR5## Crystalline forms of such salts may contain water of hydration.

Suitable esters of clavulanic acid include those notionally derived from alcohols such as methanol, ethanol, propanol, butanol, 2,2,2-trichloroethanol, 2,2,2-trifluoroethanol, benzyl alcohol, p-nitrobenzyl alcohol, phenol, acetoxymethanol, pivaloyloxymethanol, 2-dimethylaminoethanol and other conventional alcohols. Various esters of clavulanic acid are useful intermediates in certain processes for the purification of clavulanic acid. Many clavulanic acid esters are useful synergistic compounds. The activity of such esters might be due to hydrolysis of the ester to the parent acid.

When used herein the term ester includes esters notionally derived from an alcohol or thiol of the formula ROH or RSH where R is an organic residue. Suitable groups R include alkyl, alkenyl, alkynyl, aryl, arylalkyl or other similar groups any of which may be substituted if desired. In order not to increase the molecular weight to an unreasonable extent, groups R do not normally include more than 16 carbon atoms, more suitably, not more than 12 carbon atoms and most suitably, not more than 8 carbon atoms.

Preferably, the group R is notionally derived from an alcohol RCH or (less favorably) a thiol RSH which is pharmaceutically acceptable.

Suitable substituents which may be included in the group R include halogen atoms and lower alkoxyl, hydroxyl, lower acyloxyl, lower alkylamino, lower dialkylamino and like groups. The term `lower` means that the group contains up to 6 carbon atoms, and preferably up to 4 carbon atoms. Thus, for example, R may be a methyl, ethyl, n-propyl, iso-propyl, straight or branched butyl, pentyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, vinyl, allyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclohexenyl, cyclohexadienyl, methylcyclopentyl, methylcyclohexyl, benzyl, benzhydryl, phenylethyl, napthylmethyl, phenyl, naphthyl, propynyl, tolyl, 2-chlorethyl, 2,2,2-trichlorethyl, 2,2,2-tri-fluorethyl, acetylmethyl, benzoylmethyl, 2-methoxyethyl, 2-dimethylaminoethyl, 2-diethylaminoethyl, 2-piperidinoethyl, 2-morpholinoethyl, 3-dimethylaminopropyl, p-chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl, p-bromobenzyl, m-chlorobenzyl, 6-methoxynaphthyl-2-methyl, p-chlorophenyl, p-methoxyphenyl or any like group as well as those groups which are known from the penicillin or cephalosporin arts to produce esters known to be readily hydrolyzed in vivo to the parent antibiotic.

Readily hydrolyzable esters include, but are not limited to, those of the formulae (V') and (VI'): ##STR6## wherein A₁ is a hydrogen atom, alkyl, aryl or aralkyl group; A₂ is a hydrogen atom or methyl group; A₃ is an alkyl, aryl or aralkyl group; X is oxygen or sulphur; Y is oxygen or sulphur and Z is a divalent organic group. Esters of the formulae (V') and (VI') which fairly readily release the clavulanic acid into the blood stream after administration include those wherein A₁ is a hydrogen atom, A₂ is a hydrogen atom or a methyl group and A₃ is a methyl, ethyl, propyl, butyl, benzyl, or phenyl group and those wherein X is oxygen, Y is oxygen and Z is --CH₂ CH₂ --, --CH:CH--, ##STR7##

When used in conjunction with the preceding formula, the term `alkyl` includes alkyl of up to six carbon atoms; the term `aryl` includes phenyl, naphthyl or phenyl substituted by an inert substituent such as a fluorine or chlorine atom or a methyl or methoxyl group or the like; when used herein the term `aralkyl` means an alkyl group substituted by an aryl group.

Particularly suitable esters of the formulae (V') and (VI') include those of the formulae (VII') and (VIII'): ##STR8## A₄ is a hydrogen atom or a methyl group, A₅ is a methyl, t-butyl or phenyl group and A₆ is a hydrogen atom or a methoxyl group.

Many esters of clavulanic acid differ from analagous esters of penicillins or cephalosporins in that they show an enhanced tendency to hydrolyze to clavulanic acid under mild conditions. Thus, for example, simple alkyl esters such as the methyl ester slowly hydrolyze to clavulanic acid in water buffered to pH7. Esters which undergo some hydrolysis under mild conditions are included within the formula (IX): ##STR9## wherein R¹ is a hydrocarbon group of 1-9 carbon atoms optionally substituted by halogen, lower alkoxy, hydroxyl or optionally salted basic groups of the formula NR² R³ wherein R² is a hydrogen atom or a lower alkyl group, R³ is a hydrogen atom or a lower alkyl group or is attached to R² so that NR² R³ is a 5- or 6- membered ring.

When used with reference to formula (IX), the term `lower` means that the group contains 1-4 carbon atoms.

Suitably groups R¹ include alkyl and aralkyl groups optionally substituted by halogen, methoxyl, hydroxyl or salted NR² R³ groups wherein R² is a methyl or ethyl group and R³ is a methyl or ethyl group or is joined to R² so that NR² R³ is a pyrrolidine, piperidine or morpholine group.

Most suitably, alkyl groups R¹ are straight chain groups of up to 6 carbon atoms optionally substituted by one methoxyl, hydroxyl, salted NR² R³ group or one chlorine, bromine or iodine atom or by a CCl₃ or CF₃ group.

The esters of clavulanic acid of particular usefulness as synergists are those which hydrolyze in mammalian tissues, especially human blood, to yield clavulanic acid or a salt thereof because it is believed that clavulanic acid and its salts tend to be somewhat more useful antibacterial agents than the esters per se. Many of the esters of the formulae (V') - (VII'), (VIII') and (IX) are useful for this purpose.

A further group of particularly suitable esters are those useful intermediates which are readily converted to clavulanic acid or a salt thereof by chemical or biochemical techniques which are known from the penicillin or cephalosporin arts to be sufficiently mild not to degrade reactive acid-labile β-lactam rings.

Most suitably, the ester is one removable by hydrogenolysis. Conventional esters for such a process include benzyl, substituted benzyl, benzhydryl, substituted benzhydryl, trityl and the like. The benzyl ester has proved particularly useful for this purpose.

By and large, the nature of any substituent in the ester moiety is unimportant as long as it does not interfere with the hydrogenolysis reaction.

The compositions include those in a form adapted for oral, topical or parenteral use and may be used for the treatment of infection in mammals including humans.

Suitable forms of the compositions include tablets, capsules, creams, syrups, suspensions, solutions, reconstitutable powders and sterile forms suitable for injection or infusion. Such compositions may contain conventional pharmaceutically acceptable materials such as diluents, binders, colors, flavors, preservatives, disintegrants and the like in accordance with conventional pharmaceutical practice in the manner well understood by those skilled in the art of formulating antibiotics.

Injectable or infusable compositions of the clavulanic acid or its salts are particularly suitable as high tissue levels of the compound of clavulanic acid can occur after administration by injection or infusion. Thus, one preferred composition aspect comprises clavulanic acid or a salt thereof in sterile form.

Unit dose compositions comprising clavulanic acid or a salt or ester thereof adapted for oral administration form a further preferred composition.

Under certain conditions, the effectiveness of oral compositions of clavulanic acid and its salts and esters can be improved if such compositions contain a buffering agent or an enteric coating agent such that the compounds of the invention do not have prolonged contact with highly acidic gastric juice. Such buffered or enterically coated compositions may be prepared in accordance with conventional pharmaceutical practice.

The clavulanic acid or its salt or ester may be present in the composition as sole therapeutic agent or it may be present together with other therapeutic agents such as a β-lactam antibiotic. Suitable β-lactam antibiotics for inclusion in such synergistic compositions include not only those known to be highly susceptible to β-lactamases but also those which have a good degree of intrinsic resistance to β-lactamases. Thus, suitable β-lactam antibiotics for inclusion in the compositions of this invention include benzylpenicillin, phenoxymethylpenicillin, carbenicillin, methicillin, propicillin, ampicillin, amoxycillin, epicillin, ticarcillin, cyclacillin, 6-aminopenicillanic acid, 7-aminocephalosporanic acid, 7-aminodesacetoxycephalosporanic acid, cephaloridine, cephalothin, cefazolin, cephalexin, cefoxitin, cephacetrile, cephamandole, cephapirin, cephradine, cephaloglycine and other well known penicillins and cephalosporins or pro-drugs therefore such as hetacillin, metampicillin, the acetoxymethyl, pivaloyloxymethyl or phthalidyl esters of benzylpenicillin, ampicillin, amoxycillin or cephaloglycine or the phenyl, tolyl or indanyl α-esters of carbenicillin or ticarcillin or the like.

Naturally, if the penicillin or cephalosporin present in the composition is not suitable for oral administration then the composition will be adapted for parenteral administration.

When present in a pharmaceutical composition together with a β-lactam antibiotic, the ratio of clavulanic acid or its salt or ester present to β-lactam antibiotic present may be from, for example, 10:1 to 1:10 and advantageously may be from 3:1 to 1:3.

The total quantity of antibacterial agents present in any unit dosage form will normally be between 50 and 1500 mg and will usually be between 100 and 1000 mg.

Compositions may be used for the treatment of infections of inter alia, the respiratory tract, the urinary tract and soft tissues in humans.

Compositions may also be used to treat infections of domestic animals such as mastitis in cattle.

Normally between 50 and 6000 mg of the compositions will be administered each day of treatment but more usually between 500 and 300 mg of the composition of the invention will be administered per day. However for the treatment of severe systemic infections or infections of particularly intransigent organisms, higher doses may be used in accordance with clinical practice.

The exact form of the compositions will depend to some extent on the micro-organism which is being treated. For treatment of infections the compositions of this invention are normally adapted to produce a peak blood level of at least 0.1 μg/ml, more suitably at least 0.25 μg/ml, and preferably at least 1 μg/ml of clavulanic acid.

The penicillin or cephalosporin in synergistic compositions will normally be present by up to or at approximately the amount conventionally used when that penicillin or cephalosporin is the sole therapeutic agent used in the treatment of infection.

Particularly favoured compositions will contain from 150 - 1000 mg of amoxycillin, ampicillin or a pro-drug therefore and from 50 - 500 mg of clavulanic acid or a salt or in vivo hydrolyzable ester thereof and more suitably from 200 - 500 mg of amoxycillin, ampicillin or a pro-drug therefore and from 50 - 250 mg of clavulanic acid or a salt or in vivo hydrolyzable ester thereof.

The materials present in such compositions may be hydrated if required. The weights of the antibiotics in such composition are expressed on the basis of antibiotic theoretically available from the composition and not on the basis of the weight of pro-drug.

A process for the preparation of clavulanic acid and salts and esters thereof is provided which process comprises cultivating a strain of Streptomyces clavuligerus and recovering clavulanic acid or a salt thereof from the culture medium and thereafter if desired, forming the free acid or a salt or ester by methods known per se.

Preferably, Streptomyces clavuligerus ATCC 27064 or a high yielding mutant thereof is used in the process of this invention.

When used herein, the term `cultivation` means the deliberate aerobic growth of a clavulanic acid producing organism in the presence of assimilable sources of carbon, nitrogen and mineral salts. Such aerobic growth may take place in a solid or semi-solid nutritive medium, or in a liquid medium in which the nutrients are dissolved or suspended. The cultivation may take place on an aerobic surface or by submerged culture. The nutritive medium may be composed of complex nutrients or may be chemically defined. In our hands we have found media containing complex nutrients such as yeast extract, soya bean flour and the like to be particularly suitable.

The nutrient media which may be used for the cultivation of Streptomyces clavuligerus may contain, in the range 0.1 - 10% a complex organic nitrogen source such as yeast extract, corn-steep liquor, vegetable protein, seed protein, hydrolyzates of such proteins, milk protein hydrolyzates, fish and meat extracts and hydrolyzates such as peptones. Alternatively chemically defined sources of nitrogen may be used such as urea, amides, single or mixtures of common amino acids such as valine, asparagine, glutamic acid, proline and phenylalanine. Carbohydrate (0.1 - 5%) may be included in the nutrient media but glucose in certain media is undersirable having a depressing effect on the yield of the desired clavulanic acid. Starch or starch hydrolyzates such as dextrin, sucrose, lactose or other sugars or glycerol or glycerol esters may be used. The source of carbon may also be derived from vegetable oils or animal fats. Carboxylic acids and their salts can be included as a source of carbon for growth and production of β-lactamase inhibitors. A particularly suitable low cost medium is one containing soya bean flour (Arkasoy) plus dried malt distillers solubles (Scotasol) plus dextrin.

The addition of antifoam agents such as Pluronic L81 may be necessary to control foaming of certain media in fermenters.

Mineral salts such as NaCl, KCl, MgCl₂, ZnCl₂, FeCl₃, Na₂ SO₄, FeSO₄, MgSO₄ and Na⁺ or K⁺ salt of phosphoric acid may be added to the media described above particularly if chemically defined; CaCO₃ may be added as a source of Ca⁺⁺ ions or for its buffering action. Salts of trace elements such a nickel, cobalt or manganese may also be included. Vitamins may be added if desired.

When used herein the term `mutant` includes any mutant strain which arises spontaneously or through the effect of an external agent whether that agent is applied deliberately or otherwise. Suitable methods of producing mutant strains include those outlined by H.I. Adler in Techniques for the Development of Micro-Organisms in `Radiation and Radioisotopes for Industrial Micro-Organisms,` Proceedings of a Symposium, Vienna, 1973, page 241, International Atomic Energy Authority and these include:

i. Ionizing radiation (such as X- and γ-rays), uv light, uv light plus a photosensitizing agent (such as 8-methoxypsoralen), nitrous acid, hydroxylamine, pyrimidine base analogues (such as 5-bromouracil), acridines, alkylating agents (such as mustard gas, ethyl-methane sulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and

ii. Genetic techniques such as recombination, transformation, transduction, lysogenization, lysogenic conversion and selective techniques for spontaneous mutants.

Cultivation of Streptomyces clavuligerus normally takes place in the temperature range 15°-40° C, usually 20°-35° C and preferably, 25°-30° C and at a pH of between 5 and 8.5, preferably between 6 and 7.5.

The Streptomyces clavuligerus may be cultivated in the above media in glass conical flasks aerated by shaking on a rotary shaker or in baffled stainless steel fermenters stirred with vaned disc impellers and aerated with a sparger. The fermentation may also be carried out in a continuous fashion.

The starting pH of the fermentation is typically 7.0 and maximum yield of clavulanic acid obtained in 2-10 days at 20°-35° C. In a stirred stainless steel fermenter using the Arkasoy/Scotasol/Dextrin medium described above the preferred temperature is 26° C and peak yields clavulanic are obtained within 5 days.

Clavulanic acid may be extracted from culture filtrate by a variety of methods. Solvent extraction from cold culture filtrate adjusted to acid pH values and methods based on the anionic nature of the metabolite such as the use of anion exchange resins have been found to be particularly useful. The cells of the Streptomyces clavuligerus are normally first removed from the fermentation by filtration or centrifugation before such extraction procedures are commenced.

In the solvent extraction process, the culture filtrate is chilled and the pH lowered into the region of pH 2-3 by the addition of acid while thoroughly mixing with a water immiscible organic solvent such as n-butylacetate, methylisobutylketone, n-butanol or ethylacetate. The acid used to lower the pH of the medium is normally a mineral acid such as hydrochloric, sulphuric, nitric, phosphoric or the like acid. n-Butanol is a particularly suitable solvent for use in the extraction of the acidified culture filtrate. After separation of the phases by centrifugation, the β-lactamase inhibiting metabolite is back extracted from the solvent phase into aqueous sodium bicarbonate or potassium hydrogen phosphate buffer, CaCO₃ suspension or water while maintaining the pH at approximate neutrality, for example, at pH 7.0. This aqueous extract after separation of phases may be concentrated under reduced pressure and freeze dried to give a crude preparation of a salt of clavulanic acid. This preparation is stable when stored as a dry solid at -20° C.

In the anion exchange resin process, the clarified culture filtrate at an approximately neutral or slightly acid pH, for example pH 6-7, is percolated down a column of weak or strong base anion exchange resin such as Amberlite IR4B or Zerolit FFIF respectively until the resin is saturated and the β-lactamase inhibiting material emerges from the bottom. The column is then washed with water and eluted with aqueous sodium chloride. The β-lactamase inhibiting fractions are collected, bulked, desalted and freeze dried to yield a crude solid salt of clavulanic acid.

An alternative form of the extraction process is to contact the culture filtrate (usually at approximately neutral pH) containing a salt of clavulanic acid, with an organic phase in which is dissolved a water insoluble amine. Suitable organic solvents include such conventional water immiscible polar solvents as methylisobutylketone, trichloroethylene and the like. Suitable amines include secondary or tertiary amines in which one of the substituent groups is a long chain aliphatic group, for example, of 12-16 carbon atoms and the other is a tertiaryalkyl group so that the molecule is lipophilic. Amberlite LA2 has proved a successful amine. Normally the amine is used as its acid addition salt.

After this extraction process the clavulanic acid is present in the organic phase as the amine salt. The organic phase is then separated from the culture filtrate. The clavulanic acid may be back extracted into an aqueous phase by back extraction with a salt solution, preferably a concentrated solution of sodium chloride, sodium nitrate or the like. The crude salt of clavulanic acid may then be obtained by freeze drying or the like.

Other primary methods of isolation which may be used include conventional methods such as adsorption onto carbon, ion pair extraction, precipitation, salting out and molecular filtration but these methods are not usually as successful as the above described methods which are preferred.

Further purification of the crude solids obtained by methods described above may be obtained by a variety of methods but ion exchange column chromatography is particularly suitable especially when using Isopor, DeAcidite FFIP SRA64 or DEAE cellulose. The DeAcidite column is gradient eluted with aqueous solution of a salt and such sodium chloride (0-0.5M). The column of DEAE cellulose in 0.01M phosphate buffer at pH7 is eluted with a salt solution, normally a NaCl solution (0-0.2M NaCl in 0.01M phosphate buffer pH7). Active fractions may be detected by their β-lactamase inhibitory activity and their antibacterial activity against Klebsiella aerogenes in an agar diffusion assay. The fractions containing the bulk of this activity are then combined and concentrated to a small volume under vacuum. This crude preparation of the clavulanic acid salt is desalted by percolating down a column of Bio Gel P2. The active desalted material is then concentrated, mixed with ethanol and further chromatographed on a cellulose column using butanol/ethanol/water 4/1/5 v/v top phase, as solvent.

Fractions containing material which inhibited Escherichia coli β-lactamase are bulked, evaporated to dryness under vacuum, redissolved in water and freeze-dried to give a salt of clavulanic acid as a white solid.

The methods we have found most useful in detecting clavulanic acid in culture filtrates are paper chromatography and a bioautographic detection system. Clavulanic acid may be assayed by making use of its β-lactamase inhibiting activity. Thin layer chromatography may be used to detect clavulanic acid in solid preparations. These detection and assay procedures are described hereinafter.

A variation of the process for the preparation of a pure form of clavulanic acid or its salts comprises isolating an impure form of clavulanic acid or salt thereof, forming an ester of clavulanic in conventional manner, purifying the ester and thereafter regenerating clavulanic acid or a salt thereof from the ester.

The impure clavulanic acid or its salts used in this process will normally contain at least 1% by weight of the antibiotic.

Suitable esters for use in this process include those which may be cleaved by hydrogenolysis, enzymatic methods or by hydrolysis under very mild conditions.

One suitable group of esters used in this process is that of the formula (X): ##STR10## wherein A₇ is a hydrogen atom or an optionally substituted phenyl group and A₈ is an optionally substituted phenyl group.

Most suitably A₇ is a hydrogen atom or a phenyl, tolyl, chlorophenyl or methoxyphenyl group and A₈ is a phenyl, tolyl, chlorophenyl or methoxyphenyl group.

Preferably A₇ is a hydrogen atom and A₈ is a phenyl group.

The esters of formula (X) may be cleaved by hydrogenolysis to yield clavulanic acid or a salt thereof.

Other groups of esters which may be used in this process include those of formulae (V') and (VI') as hereinbefore described. Such esters may be converted to salts of clavulanic acid by mild alkaline hydrolysis, for example, at pH 7.5.

The impure form of clavulanic acid or salt thereof which is to be purified in this process may be in the form of a solid or solution which will usually also contain considerable quantities of organic or inorganic impurities.

The clavulanic acid or salt thereof may be converted into an ester by the esterification reactions referred to hereinafter. The preferred method of forming the required ester of clavulanic acid is by the reaction of a salt of clavulanic acid with an esterifying agent such as a reactive halide, sulphonate ester or the like as hereinafter described. Such reactions are frequently carried out in an organic solvent of high dielectric constant such as dimethylformamide, dimethylformamide/acetone, dimethylsulphoxide, N-methylacetamide, hexamethylphosphoramide and the like.

If desired, the salt of clavulanic acid may be dissolved in the solvent in conventional manner or it may be bound to a polymeric support. Suitable supports for use in this process include strong base anion exchange resins, especially those possessing a macroreticular nature which permits the use of non-aqueous solvent systems.

Amerblyst A26 is suitable for this purpose. The clavulanic acid salt may be adsorbed onto the resin from the culture filtrate and the resin then suspended in dimethylformamide containing sodium iodide or alternatively eluted columnwise with a solution of sodium iodide in dimethylformamide or in a mixture of dimethylformamide and acetone.

Once formed, the impure ester of clavulanic acid is normally purified chromatographically. In such procedures the ester is normally dissolved in an organic solvent such as ethylacetate, methylene chloride, chloroform, cyclohexane or similar solvents. The solid phase used in the chromatographic process is normally an inert material such as silica gel or chromatographically similar materials.

The fractions emerging from the column may be tested for the presence of the clavulanic acid by making use of its synergistic properties. Active fractions are normally combined and the organic solvent evaporated off under reduced pressure.

The ester resulting from this process is generally of acceptable purity, but the material may be rechromatographed if desired.

This purified ester of clavulanic acid may be converted to clavulanic acid or a salt thereof by the before mentioned methods.

A particularly suitable method of obtaining clavulanic acid or its salt is by hydrogenation of a compound of the formula (X) as hereinbefore described. Such reactions normally take place in the presence of a transition metal catalyst using low or medium pressures of hydrogen. The reaction may be carried out at high, ambient or depressed temperatures, for example at 0°-100° C. Particularly suitable reaction conditions for such hydrogenations will use a slightly superatmospheric pressure of hydrogen at an approximately ambient (12°-20 C) temperature. The reaction may be carried out in conventional solvents such as lower alkanols, for example, ethanol. We have found that a particularly suitable catalyst is palladium on charcoal.

If the hydrogenation is carried out in the presence of a base, then a salt of clavulanic acid is produced; for example, the sodium or potassium salts result if the reaction is carried out in the presence of sodium or potassium hydrogen carbonate.

The clavulanic acid or salt thereof resulting from such reactions is generally of good purity.

Esters or clavulanic acid may be prepared by the esterification of clavulanic acid or a salt thereof by conventional methods.

Suitable methods of ester formation include (a) reaction of a salt of the acid of clavulanic acid with a compound of the formula Q - R where Q is a readily displaceable group and R is an organic group; (b) the reaction of clavulanic acid with a diazoalkane and (c) the reaction of clavulanic acid with an alcohol ROH in the presence of a condensation promoting agent such as carbodiimide or the like.

Suitable salts of clavulanic acid which may be reacted with compounds R - Q include alkali metal salts such as the sodium or potassium salts or other conventional salts such as the silver salt.

Suitable groups Q include those atoms or groups known to be displaceable by carboxylate anions and include chlorine, bromine and iodine atoms, sulphonic acid esters such as the O.SO₂ CH₃ or O.SO₂ C₆ H₄ CH₃ groups, active ester groups such as the O.CO.H or O.CO.CF₃ group and other conventional groups displaceable by nucleophiles.

The preceding reaction is normally carried out in an organic solvent of relatively high dielectric constant such as dimethylformamide, acetone, dioxane, tetrahydrofuran or the like and at a non-extreme temperature such as -5° C to 100° C, more usually +5° C to 30° C, for example at ambient temperature.

The reaction of clavulanic acid with a diazoalkane is a mild method of making alkyl, aralkyl or similar esters. The diazotization reaction may be performed under conventional reaction conditions, for example at a non-extreme temperature and in a conventional solvent. Such reactions are normally carried out at between about -5° C and 100° C, more usually from 5° C to 30° C, for example at ambient temperature. Suitable solvents for this reaction include lower alkanols such as methanol and ethanol and solvents such as tetrahydrofuran, dioxane and the like. Ethanol has proved a particularly useful solvent for this reaction.

The reaction of clavulanic acid with an alcohol in the presence of a condensation promoting agent will normally take place in an inert organic solvent of relatively high dielectric constant such as acetonitrile. This reaction is usually carried out at an ambient or depressed temperature, for example at -10° C to +22° C, more usually -5° C to +18° C, for example initially at 0° C and thereafter gradually warming to about 15° C. The condensation promoting agent used is normally one which removes water from the reaction mixture. Suitable agents include carbodiimides, carbodiimidazoles or equivalent reagents. Dicyclohexylcarbodiimide has proved to be a particularly suitable condensation promoting agent for use in this process. In order to minimize self-condensation of the clavulanic acid, this reaction is usually carried out in the presence of a considerable excess of the alcohol.

Other suitable methods of ester formation include (d) removal of the elements of carbon dioxide from a compound of the formula (XI) ##STR11## wherein R⁴ is an inert organic group; and (e) reaction of a compound of the formula (XI) with an alcohol ROH (or less favorably with a thiol RSH).

The elements of carbon dioxide may be removed from the compound of formula (XI) spontaneously during its preparation or, alternatively, by heating the compound of the formula (XI) in an inert solvent. Suitable inert solvents include ether solvents such as diethylether, tetrahydrofuran, dioxane, and the like. In many cases the compound of the formula (XI) decomposes spontaneously even at a depressed temperature, for example, at -5° C, to yield an ester of the formula ##STR12## wherein R⁴ is a inert group within the definition of R.

When the compound of the formula (XI) is to be reacted with an alcohol (or less favorably with a thiol) then this reaction is normally carried out in an inert solvent such as an ether solvent in the presence of an excess of the alcohol (or thiol) in order to prevent self-condensation of the clavulanic acid derivative.

The methods of esterification are not, in general, as useful as those involving reaction of a salt of clavulanic acid with R-Cl as hereinbefore described.

The compound of the formula (XI) may be prepared by the reaction of a salt of clavulanic acid with Cl.CO.O.R⁴ or the chemical equivalent thereof. Normally this reaction is carried out at a depressed temperature, for example, at a temperature not greater than 5° C, and in an inert solvent, for example diethylether, tetrahydrofuran, dioxane and the like. Most suitably the salt of clavulanic acid used in this reaction is a lipophilic salt so that it will dissolve in the solvent although if desired the less favorable sodium salt may be employed by suspending it in the reaction medium.

DESCRIPTION 1 ASSAY SUITABLE FOR DETECTION OF CLAVULANIC ACID Principle of the Assay

Solutions containing clavulanic acid (culture filtrate, samples from isolation procedure and the like) are incubated for 15 minutes with a β-lactamase preparation in 0.05M phosphate buffer at pH 7 and 37° C. During this time, enzyme inhibition or inactivation occurs. Substrate (benzylpenicillin) is then added and incubation continued for 30 minutes at 37° C. The amount of enzymic degradation of the substrate to penicilloic acid is determined by the hydroxylamine assay for penicillin. The amount of β-lactamase used is such as to give 75% hydrolysis of the benzylpenicillin in 30 minutes at 37° C.

The extent of hydrolysis is a reflection of the amount of enzyme remaining uninhibited. The results are expressed as percent inhibition of the enzyme activity by a given dilution of the clavulanic acid-containing solution (e.g. culture filtrate) or the concentration of clavulanic acid (μg/ml) giving 50% inhibition of the enzyme under the above stated conditions (I₅₀).

β-lactamase Enzyme

The β-lactamase produced by Escherichia coli JT4 is used as an enzyme. This culture is an ampicillin resistant strain and owes its resistance to the production of an R-factor controlled β-lactamase. Other similar R-factor controlled β-lactamases may be used if desired.

The culture maintained on nutrient agar slopes, is inoculated into 400 ml. of sterile Tryptone medium contained in a 2 liter conical flask. This medium has the following composition Tryptone (Oxoid) 32 g/l, yeast extract (Oxoid) 20 g/l, NaCl 5 g/l and CaCl₂ 6H₂ O 2.2 g/l. The final pH was adjusted to 7.4 with dilute NaOH. The flask is shaken at 25° C for 20 hours on a rotary shaker at 240 r.p.m.

The bacterial cells are collected by centrifugation, washed with 0.05M phosphate buffer pH 7 (resuspended and centrifuged) and resuspended in water to give cell concentration 25 times that in the cultivation medium. This cell suspension was then disrupted in an MSE ultrasonic disintegrator at 4° C. The cell debris was removed by centrifugation and aliquots of the supernatant stored deep frozen. For use in the assay procedure, the supernatant is diluted in 0.05M phosphate buffer until it gives about 75% hydrolysis of a 1 mg/ml. solution of benzylpenicillin in 30 minutes at 37° C.

Assay Procedure

Suitable dilutions of the inhibitor preparation and β-lactamase solution are mixed and incubated at 37° C for 15 minutes (Test). A control with buffer in place of inhibitor preparation is also incubated. Benzylpenicillin solution (substrate) is then added to test and control mixtures, incubation continued for a further 30 minutes at 37° C. The residual benzylpenicillin in each mixture is then estimated using the hydroxylamine assay as described by Batchelor et al, Proc. Roy. Soc., B 154, 498 (1961). 6 ml. of hydroxylamine reagent are added to all tests, controls and blanks and are allowed to react for 10 minutes at room temperature prior to the addition of 2 ml of ferric ammonium sulphate reagent. The absorption of the final solutions is measured in an E.E.L. Colorimeter or a Spectrophotometer at 490 nm against the reagent blank. The composition of the reactions, tests and blanks prior to the hydroxylamine assay are as follows:

    ______________________________________                                         Components            Benzyl-                                                  (all dissolved in or  penicillin                                                                               Con-  Reagent                                  diluted with 0.005M   Blank     trol  Blank                                    pH 7 phosphate buffer)                                                                        Test   ml.       ml.   ml.                                      ______________________________________                                         Escherichia coli                                                               β-lactamase solution                                                                     1.9    0.0       1.9   1.9                                      Inhibitor solution                                                                            0.1    0.0       0.0   0.0                                      Benzylpenicillin                                                               5mg/ml.        0.5    0.5       0.5   0.0                                      0.005M pH 7 phosphate                                                          buffer         0.0    2.0       0.1   0.6                                      ______________________________________                                    

Calculation of Results

The percentage inhibition of the β-lactamase is calculated as follows

Absorption of benzylpenicillin blank minus absorption of control (uninhibited reaction) = x

Absorption of test (inhibited reaction) minus absorption of control (uninhibited reaction) = y

    % inhibition = y/x × 100

To obtain the I₅₀ value, the inhibitor preparation is diluted until 50% inhibition of the β-lactamase inactivation of benzylpenicillin is obtained in the above procedure.

DESCRIPTION 2 PAPER CHROMATOGRAPHIC DETECTION OF CLAVULANIC ACID

Culture filtrate and a reference solution of clavulanic acid (250 μg/ml partially purified preparation), are spotted (20 μl/origin) onto Whatman No. 1 paper strips 1 cm. wide. The chromatograms are run by descending chromatography for 16 hours at 5° C using n-butanol/isopropanol/water, 7/7/6 v/v as solvent. The strips are dried at 40° C and laid on agar plates containing 6 μg/ml benzylpenicillin and seeded with a β-lactamase producing strain of Klebsiella aerogenes (synergism system). The plates are incubated overnight at 30° C and clavulanic acid revealed as a zone of inhibited growth. The R_(f) value of the zone was 0.46. The 6 μg/ml benzylpenicillin alone is below the concentration required to kill the Klebsiella aerogenes but in the presence of a β-lactamase inhibitor, this concentration becomes toxic, that is to say there is synergism.

Use of the above synergism system enables clavulanic acid to be detected at concentrations below those at which it shows antibacterial activity.

DESCRIPTION 3 THIN LAYER CHROMATOGRAPHIC DETECTION OF CLAVULANIC ACID SODIUM SALT

Solutions of clavulanic acid sodium salt preparations are spotted (5 μl of 1mg/ml) onto glass plates coated with a 0.25 mm layer of silica gel (F254) as supplied by E. Merck, Darmstadt, Germany. The chromatograms are run at 22° C using the top phase of the mixture n-butanol/ethanol/water 4/1/5 v/v. The chromatogram plates are dried at 40° C and clavulanic acid sodium salt located by bioautography on agar plates containing 6 μg/ml. benzylpenicillin and seeded with Klebsiella aerogenes (synergism system - see section on paper chromatography above). The agar surface is covered by a fine filter cloth before laying the TLC plate onto it. After allowing 15-30 minutes for wetting and diffusion, the TLC plate is lifted off with the aid of the filter cloth and the agar plate incubated overnight at 30° C to reveal the zones of inhibited growth. The R_(f) value of clavulanic acid sodium salt in the above solvent is approximately 0.37. Two spray reagents, Ehrlich and triphenyltetrazolium chloride are also used to reveal the clavulanic acid sodium salt zone. The former reagent consists of 300 mg of p-dimethylaminobenzaldehyde dissolved in 9 ml. of ethyl alcohol, 54 ml. of n-butanol and 9 ml of concentrated HCl. On heating the sprayed TLC plate at 120° C for 1-2 minutes, clavulanic acid sodium salt appears as a pink spot. The triphenyltetrazolium chloride reagent consists of a mixture of 1 volume of a 4% solution of this compound in methanol with 1 volume of methanolic sodium hydroxide. After spraying, the TLC plates are heated at 80° C. Clavulanic acid sodium salt appears as a red spot on a white background.

The following Examples are illustrative:

EXAMPLE 1 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

Streptomyces clavuligerus was cultivated at 26° C on agar slopes containing 1% Yeatex (yeast extract), 1% glucose and 2% Oxoid agar No. 3, pH 6.8. A sterile loop was used to transfer mycelium and spores from the slope into 100 ml of a liquid medium in a 500 ml Ehrlenmeyer flask.

The liquid medium had the following composition:

    ______________________________________                                         Oxoid Malt Extract     10 g/l                                                  Oxoid Bactericlogical Peptone                                                                         10 g/l                                                  Glycerol               20 g/l                                                  Tap water              1 liter                                                 ______________________________________                                    

The medium was adjusted to pH 7.0 with sodium hydroxide solution and 100 ml. volumes dispensed into flasks which were closed with foam plugs prior to autoclaving at 15 lb/sq. in. for 20 minutes. An inoculated seed flask was shaken for 3 days at 26° C on a rotary shaker with 2 inch throw and a speed of 240 r.p.m. Production stage flasks containing the liquid medium described above were inoculated with 5% vegetative inoculum and grown under the same conditions as the seed flask. Samples of culture filtrate were assayed for inhibitor action against the β-lactamase of Escherichia coli JT4. Optimum activity was obtained after 3 days. The results are shown in Table 1. A zone of clavulanic acid at R_(f) 0.46 was seen when the culture filtrate was examined by the paper chromatographic method previously described. The increase in size of the zone paralleled the increase in the β-lactamase inhibitor assay.

Streptomyces clavuligerus was also cultivated in 2 liter shaken flasks containing 400 mls. of medium (Production stage) using the same medium and cultural conditions as described earlier in this Example. In these larger vessels, growth of the organism was slower and optimum β-lactamase inhibitory activity was achieved 7-9 days after inoculation with the vegetative seed. The results are also shown in Table 1.

                  TABLE 1                                                          ______________________________________                                         β-Lactamase                                                               Inhibiting Activity of Streptomyces clavuligerus                               Grown in 500 ml. and 2000ml. Flasks                                                     % Inhibition of Escherichia coli                                      Fermentation                                                                            β-lactamase at a final dilution of                               Time     1/2500 of culture filtrate                                            (Days)   500 ml. Shaken Flask                                                                           2000 ml. Shaken Flask                                 ______________________________________                                         1        15              --                                                    2        30              --                                                    3        55              --                                                    4        50              10                                                    5        51              21                                                    6        57              36                                                    7        --              51                                                    8        --              53                                                    9        --              50                                                    ______________________________________                                    

EXAMPLE 2 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

A seed flask prepared as in Example 1 was used to inoculate 500 ml. conical flasks containing 100 ml. aliquots of the following medium in deionized water:

    ______________________________________                                         Soluble Starch     2%         w/v                                              Glycerol           0.3%       w/v                                              Scotasol           0.1%       w/v                                              Arkasoy            1%         w/v                                              FeSO.sub.4 · 7H.sub.2 O                                                                  0.01%      w/v                                              ______________________________________                                    

The medium was sterilized by autoclaving at 15 p.s.i. for 20 minutes and inoculated by the addition of the 5% vegetative seed stage. The flasks were shaken at 26° C on a rotary shaker as in Example 1. Optimum titre of clavulanic acid was achieved between 3-5 days. A dilution of 1/2500 of the culture filtrate gave 60% inhibition in the β-lactamase inhibition assay. A zone of clavulanic acid was seen at R_(f) 0.46 when using the paper chromatographic (bioautographic) method previously described. This zone increased in size in parallel with the increase of the activity in the β- lactamase inhibitor assay.

[Soluble starch supplied by British Drug Houses Ltd., Poole, U.K.;

Scotasol is dried distillers solubles supplied by Thomas Borthwich Ltd., 60 Wellington Street, Glasgow, UK;

Arkasoy is soya bean flour supplied by British Arkady Co., Old Trafford, Manchester, UK].

EXAMPLE 3 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

A seed flask as produced in Example 1 was used to inoculate 500 ml. conical flasks containing 100 ml aliquots of the following medium prepared in deionized water and sterilized as previously described. The inoculum level was 5%.

    ______________________________________                                         Dextrin            2%         w/v                                              Arkasoy            1%         w/v                                              Scotasol           0.1%       w/v                                              FeSO.sub.4 7H.sub.2 O                                                                             0.01%      w/v                                              ______________________________________                                    

The inoculated flasks were shaken at 26° C. Optimum β-lactamase inhibitory activity was achieved between 3-5 days. The activity was similar to that achieved in Example 2.

[Dextrin is supplied by C P C (UK) Ltd., Trafford Park, Manchester, UK]

EXAMPLE 4 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

The seed stage as described in Example 1 was used to inoculate 500 ml. conical flasks containing the following medium prepared in deionized water.

    ______________________________________                                         Dextrose           1%         w/v                                              Soyabean Meal      1%         w/v                                              Scotasol           0.05%      w/v                                              CaCO.sub.3         1%         w/v                                              ______________________________________                                    

These flasks were treated exactly as in previous Examples and cultured under identical conditions. β-lactamase inhibitory activity was produced between 3-5 days. Culture filtrate at a final dilution of 1/2500 gave 35-45% inhibition in the β-lactamase inhibition assay.

EXAMPLE 5 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

β-lactamase inhibitory activity attributable to clavulanic acid was produced using the following medium with identical seed stage and cultivation conditions to Example 1.

    ______________________________________                                         Glycerol           2%         w/v                                              Soyabean Meal      1.5%       w/v                                              Mg SO.sub.4        0.1%       w/v                                              K.sub.2 HPO.sub.4  0.1%       w/v                                              ______________________________________                                    

Medium prepared in deionized water.

β-lactamase inhibitory activity reached a maximum level between 3-5 days and was of a similar order to that produced in Example 4.

EXAMPLE 6 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

The following medium produced clavulanic acid when using the conditions and vegetative seed inoculum as described in Example 1.

    ______________________________________                                         Glucose             2%                                                         Lab Lemco (Oxoid)   1%                                                         Oxoid Yeast Extract                                                                               0.3%                                                        CaCO.sub.3         0.3%                                                        ______________________________________                                    

Medium prepared in deionized water.

Optimum titres were achieved in 3-5 days and a 1/2500 dilution of the culture filtrate gave 35-45% inhibition in the β-lactamase enzyme inhibition assay.

EXAMPLE 7 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

As in Examples 4, 5 and 6 the following medium produced 35-45% inhibition (1/2500 dilution) in the β-lactamase assay at the optimum titre which is reached 3-5 days after inoculation. All conditions were as previously described.

    ______________________________________                                         Glucose            2%         w/v                                              Arkasoy            1%         w/v                                              CaCO.sub.3         0.02%      w/v                                              CoCl.sub.2 . 6H.sub.2 O                                                                           0.0001%    w/v                                              ______________________________________                                    

Medium prepared in deionized water.

EXAMPLE 8 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

The following production stage medium when used under standard cultivation conditions as described in previous Examples produced 20-30% inhibition at 1/2500 dilution in the β-lactamase assay between 3-5 days after inoculation. Using the paper chromatographic method previously described, a zone of clavulanic acid was seen at R_(f) 0.46 when culture filtrate was examined.

    ______________________________________                                         Scotasol               2%                                                      Oxoid Yeast Extract    1%                                                      Medium prepared in tap water                                                   Final pH 7.0                                                                   ______________________________________                                    

EXAMPLE 9 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

Under standard cultivation conditions, the following medium produced clavulanic acid 3-5 days after inoculation with the vegetative seed. A 1/2500 dilution of the culture gave 20-30% inhibition in the β-lactamase inhibition assay.

    ______________________________________                                                            g/l                                                         ______________________________________                                         Glycerol             15                                                        Sucrose              20                                                        Proline              2.5                                                       Monosodium Glutamate 1.5                                                       NaCl                 5.0                                                       K.sub.2 HPO.sub.4    2.0                                                       CaCl.sub.2           0.4                                                       MnCl.sub.2 4H.sub.2 O                                                                               0.1                                                       FeCl.sub.3 6H.sub.2 O                                                                               0.1                                                       ZnCl.sub.2           0.05                                                      MgSO.sub.4 7H.sub.2 O                                                                               1.0                                                       ______________________________________                                    

Medium prepared in deionized water. Final pH 7.1.

EXAMPLE 10 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

A stock Yeatex/glucose agar slope was used to inoculate a Yeatex/glucose agar slope in a Roux bottle by making a mycelium/spore suspension in sterile water. The Roux bottle slope was incubated at 26° C for 10 days. To this slope 100 mls. of sterile water was added and a mycelial suspension prepared. This was used to inoculate 50 liter of steam sterilized seed medium of the following composition in tap water.

    ______________________________________                                         Oxoid Malt Extract    1%       w/v                                             Oxoid Bacteriological Peptone                                                                        1%       w/v                                             Glycerol              1%       w/v                                             10% Pluronic L81 Antifoam in                                                   Soyabean Oil          0.05%    w/v                                             ______________________________________                                    

[Pluronic supplied by Jacobs and Van den Berg UK Ltd., 231 The Vale, London, W3 containing a polypropylene-polyethylene block polymer, and Soyabean Oil supplied by British Oil and Cake Mills Ltd., Stoneferry Road, Hull, U.K.].

The medium was contained in a 90 liter stainless steel baffled fermenter, agitated by a 5 inches vaned disc impeller at 240 r.p.m. Sterile air was supplied at 50 l/min and the tank incubated at 26° C.

After 72 hours, the seed fermenter was used to inoculate 150 liter of the same medium using a 5% v/v addition by sterile transfer. This production stage medium was contained in a 300 L stainless steel, fully baffled fermenter agitated by a 81/2 inches vaned disc impeller at 210 r.p.m. Sterile air was supplied at 150 l/min. The fermentation was maintained at 26° C. Antifoam was added when required in 10 ml shots (10% Pluronic L81 in soyabean oil). Samples were removed for β-lactamase inhibition assay at regular intervals. The fermenter was harvested between 4-5 days at the optimum level of β-lactamase inhibitory activity (Table 2).

                  TABLE 2                                                          ______________________________________                                         β-Lactamase Inhibitory Activity of Samples of                             Culture Filtrate taken from a 300 liter                                        Fermentation of Streptomyces Clavuligerus                                      Fermentation % Inhibition in β-lactamase                                  Time         Inhibition Assay at a Final                                       (days)       Dilution of 1/2500                                                ______________________________________                                         1.0          12                                                                1.5          20                                                                2.0          31                                                                2.5          36                                                                3.0          50                                                                3.5          54                                                                4.0          51                                                                4.5          56                                                                5.0          55                                                                ______________________________________                                    

EXAMPLE 11 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

The seed fermenter was run exactly as described in Example 10 using the same medium.

After 72 hours, the seed fermenter was used to give a 5% v/v vegetative inoculum into a 300 liter stainless steel fully baffled fermenter containing 150 liter of steam sterilized medium agitated by an 81/2 inch vaned disc impeller at 210 r.p.m. Sterile air was supplied at 150 l/min. The fermentation was maintained at 26° C. Antifoam was added when required in 10 ml. shots (10% Pluronic L81 in soya bean oil).

The medium used in the production stage was as described in Example 3 with the addition of 0.05% v/v of 10% Pluronic L81/soyabean oil antifoam prior to sterilization.

The β-lactamase inhibitory activity of fermentation samples was similar to those of Example 10 (see Table 2). Paper chromatographic examination revealed a zone of clavulanic acid at R_(f) 0.46 using the bioautographic (synergism) method previously described. The size of the clavulanic acid zone increased in parallel with the increase in the β-lactamase inhibitor assay.

EXAMPLE 12 CULTIVATION OF STREPTYMYCES CLAVULIGERUS

100 mls of sterile water was added to a sporing culture which had been grown on Bennetts agar in a Roux bottle for 10 days at 26° C. A mycelium/spore suspension was produced and used to inoculate 75 liters of steam sterilized medium of the following composition in tap water.

    ______________________________________                                         Dextrin             2%        W/V                                              Arkasoy `50`        1%        W/V                                              10% Pluronic L81    0.03%     V/V                                              in soyabean oil                                                                ______________________________________                                    

The pH of the medium was adjusted to 7.0.

The medium was contained in a 100 liter stainless steel baffled fermenter, agitated by a 71/2 inches vaned disc impeller at 140 rpm. Sterile air was supplied at 75 l/minute and the tank incubated for 72 hours at 26° C.

The contents of the seed fermenter were used to inoculate 1500 liters of steam sterilized medium of the following composition in tap water.

    ______________________________________                                         Arkasoy `50`         1.5% W/V                                                  Glycerol             1.0% W/V                                                  KH.sub.2 PO.sub.4    0.1% W/V                                                  10% Pluronic L81     0.2% V/V                                                  in soyabean oil                                                                ______________________________________                                    

The pH of the medium was adjusted to 7.0.

The medium was contained in a 2000 liter stainless steel fully baffled fermenter agitated by two 19 inches vaned disc impellers at 106 r.p.m.

Sterile air was supplied at 1200 liters per minute. Antifoam was added in 25 ml amounts as required. (10% Pluronic L81 in soyabean oil). The fermentation was controlled at 26° C until a maximum yield of clavulanic acid was obtained between 3 - 5 days when 200 - 300 μg/ml of clavulanic acid were produced.

EXAMPLE 13 CULTIVATION OF STREPTOMYCES CLAVULIGERUS

Inoculum was produced in a seed flask as previously described, but using the medium described in Example 3 (with pH of the medium adjusted to 7.0). This was used to inoculate 500 ml conical flasks containing 100 ml aliquots of the following medium prepared in deionized water and sterilized. The inoculum level was 5%.

    ______________________________________                                         Prichem P224       1%         W/V                                              Arkasoy `50`       1.5%       W/V                                              KH.sub.2 PO.sub.4  0.1%       W/V                                              ______________________________________                                    

The pH of the medium was adjusted to 7.0.

The inoculated flasks were shaken at 26° C and optimum β-lactamase inhibitory activity was achieved between 3 - 5 days. Levels of 300 - 500 μg/ml of clavulanic acid were achieved.

Prichem P224 is a triglyceride supplied by Prices Limited, Bromborough, Bebington, Wirral, Cheshire, U.K.

EXAMPLE 14 ISOLATION OF CRUDE CLAVULANIC ACID SODIUM SALT

Harvested culture liquor produced as described in Example 10 was clarified by continuous-flow centrifugation and the mycelium discarded. From 150 liter of fermentation liquor 120 liter of clarified culture fluid was obtained. This filtrate gave 58% inhibition in the β-lactamase inhibition assay at 1/2500. The filtrate was chilled to 5° C and 40 liter of n-butanol added. The mixture was stirred and 25% H₂ SO₄ added until the pH was 2.0. The acidified mixture was stirred for a further 10 mins. before separating the phases by centrifugation. The aqueous phase was discarded. To the n-butanol extract 0.5% of Norit GSX carbon was added and the mixture stirred for 15 minutes. The carbon was discarded after removal by filtration using a diatomaceous earth as a filter aid. To the n-butanol a one-fourth volume of deionized water was added and the mixture stirred while adding 20% NaOH solution until the pH had equilibrated at 7.0. The phases were separated by centrifugation and the n-butanol phase discarded. The aqueous phase was concentrated under reduced vacuum to 800 ml. and then freeze-dried. This yielded 35g. of a crude solid preparation of clavulanic acid with an I₅₀ of 1.3 μg/ml in the β-lactamase inhibition assay. This solid preparation was stored dry at -20° C while awaiting further purification.

EXAMPLE 15 ISOLATION OF CRUDE CLAVULANIC ACID SODIUM SALT

One liter of culture filtrate giving 53% inhibition at 1/2500 in the β-lactamase inhibition assay and obtained as described in Example 12 was percolated down a 1 inch diameter x 6 inch column of Permutit Isopore resin FF 1P (SRA 62) in the Cl⁻ form [supplied by Permutit Co. Ltd., 632-652 London Road, Isleworth, Middlesex, U.K.]. The culture filtrate was followed by 300 ml of distilled water to wash the column. Elution of the active β-lactamase inhibitor was achieved with 0.2M NaCl solution. Fractions (20 ml.) were collected and assayed at a 1/2500 final dilution in the β-lactamase inhibition assay. Active fractions were combined and concentrated under vacuum to 20 ml. This solution was desalted by gel exclusion chromatography on a Biorad Biogel P2 column 11/2 inches in diameter with a gel bed of 16 inches and eluted with 1% n-butanol in water. [Biogel P2 is supplied by Bio Rad Laboratories, 32nd and Griffin Ave., Richmond, California, U.S.A.]. The active fractions, as determined by the β-lactamase inhibition assay, were combined. Sodium chloride eluted after clavulanic acid and was detected using silver nitrate solution. The combined active fractions were concentrated and freeze dried.

One liter of culture filtrate after the above treatment yielded 0.45g. of a crude solid preparation of clavulanic acid having an I₅₀ of 0.92 μg/ml.

This solid was stored at -20° C while awaiting further purification.

EXAMPLE 16 ISOLATION OF CRUDE CLAVULANIC SODIUM SALT

Culture filtrate containing 300 μg/ml of clavulanic acid is acidified using an in-line mixer system, extracted with n-butanol and clavulanic acid is back extracted into water at neutral pH.

Chilled culture filtrate (5° - 10° C) was pumped to an in-line mixer at the inlet of which, enough 6% (v/v) nitric acid was added to maintain an outlet pH of 2.0 ± 0.1. The acidified filtrate was passed at 4.1/min through a glycolcooled-plate heat exchanger (A.P.V. Ltd.) to maintain a temperature between 2° - 5°. The pH was monitored in a flow-cell before passing into a three-stage counter-current separator (Westfalia Separator Ltd., Model EG 1006).

Chilled water saturated n-butanol (at about 5° C) was pumped at 3 l/min into the counter-current separator.

The aqueous outlet from the counter-current separator was run to waste. Entrained water was removed from the butanol outflow of the counter current separator using a liquid/liquid centrifugal separator. (Alfa Laval Ltd. Model 3024X - G). The butanol was collected in a stainless steel vessel fitted with a cooling jacket in which it was stored at about 5° C.

From the vessel, 40 l aliquots were removed and thoroughly mixed with 2 l of chilled water (5° C), saturated with n-butanol. The pH of this mixture was adjusted to pH 6.8 ± 0.1 using 20% sodium hydroxide solution.

This aqueous extract/butanol mixture was fed to a liquid/liquid centrifugal separator (Sharples Centrifuge Ltd. Model M35PY - 5 PH) at a pumped rate of 2 l/min.

From 1800 l of culture filtrate, 90 l of aqueous phase was recovered, containing 39% of the clavulanic acid present in the culture filtrate.

15 l of the aqueous extract was adjusted from 2%, to 8%, total solids by the addition of 60 g sodium chloride per liter, and spray-dried (Anhydro, Copenhagen, Type Lab S l). The conditions used were: Feed rate 2 l/hr Atomizer voltage 170 v; Heater setting 6 - 7; Inlet temp 150° C; Outlet temp 80° C.

The dried product, total weight 1 kg., contained 62% of the clavulanic acid present in the feedstock.

The remaining 75 l of aqueous extract was concentrated by ultrafiltration (De Danske Sukkerfabrikker. Laboratory Module, Membrane Type 900). The operating procedure was to re-circulate the retentate from a stainless steel tank, fitted with a cooling system, with the outlet valve set so as to give a differential pressure across the 40 membranes of 25 atmospheres. The temperature was maintained at 2° - 5° C and the pH at 6.8 ± 0.1 by addition of 2N hydrochloric acid, as necessary. The volume was reduced to 34 l which contained 72% of the clavulanic acid present in the feedstock.

The aqueous concentrate was stored at about 5° C, adjusted to 8% solids, and spray dried as above. The dried material contained 75% of the clavulanic acid present in the feedstock to the spray drier.

The total spray-dried product, from the 90 l of aqueous extract contained 69.4 g of clavulanic acid which was 72% of the clavulanic acid in the spray-drying feedstock and 21% of the clavulanic acid present in the 1800 l of culture filtrate.

EXAMPLE 17 PARTIAL PURIFICATION OF CRUDE CLAVULANIC ACID

Crude clavulanic acid preparations obtained as described in Example 15 were purified by ion exchange chromatography. Eighteen grams of material prepared as described in Example 15 having an I₅₀ value of 1.3 βg/ml (final concentration) were dissolved in 25 ml. of distilled water and applied to a 11/2 × 16 inches bed of Permutit FF 1P (SRA 62) resin in the chloride form. The column was eluted with a sodium chloride gradient formed by gravity feeding 0.5M sodium chloride into a mixing reservoir containing 1 liter of distilled water which in turn fed the chromatographic column. 10 ml. cuts were collected and β-lactamase inhibitory activity assayed using a 1/2500 dilution of the fractions. Activity was eluted after a main band of color between fractions 24 and 30. The active fractions were combined and concentrated to 30 ml.

This solution was desalted using a 2 × 18 bed of Biorad Biogel P2 and eluting with 1% n-butanol in water. The 20 ml. fractions were assayed for clavulanic acid content using the β-lactamase inhibition assay. The fractions were also spotted onto paper strips and sprayed with either the Ehrlich or the triphenyltetrazolium spray reagents described in Description 3. β-lactamase inhibitory activity correlated with the pink or red spots respectively produced by these reagents. Active cuts were combined, excluding those containing sodium chloride and concentrated under vacuum to dryness. This yielded 520 mg. of partially purified clavulanic acid sodium salt with an I₅₀ of 0.2 μg/ml in the standard β-lactamase inhibitor assay.

Thin layer chromatography (silica gel) of this clavulanic acid preparation gave the following R_(f) values: n-butanol/ethanol/water 4:1:5 v/v top phase R_(f) 0.37; n-butanol/acetic acid/water 12:3:5 v/v R_(f) 0.44; isopropanol/water 7:3 v/v R_(f) 0.78. The zones were detected by spraying with Ehrlich's reagent. 6-Aminopenicillanic run as a marker and detected with the same spray had R_(f) values of 0.38; 0.39 and 0.77 respectively.

EXAMPLE 18 PARTIAL PURIFICATION OF CLAVULANIC ACID SODIUM SALT

Culture filtrate produced as described in Example 12 was solvent extracted as in Example 14 to give a solid preparation which was further purified by ion exchange chromatography using Whatman diethylaminoethyl cellulose DE 52. This solid (10g. ) was dissolved in 20 ml. of distilled water and applied to a 11/2 × 20 inches column of DE 52 cellulose previously equilibrated with 0.01M sodium phosphate buffer pH 7.5. The column was eluted with a NaCl gradient. 0.1M NaCl in 0.01M sodium phosphate buffer pH 7.5 was fed into a mixing chamber containing 1 liter of 0.01M phosphate buffer pH 7.5 which in turn was connected to the column. Fractions (10 ml. ) were collected and these were assayed for β-lactamase inhibitory activity at a dilution of 1/2500. The fractions were also examined for antibacterial activity by the hole-in-plate assay method using nutrient agar plates seeded with Klebsiella aerogenes. The fractions having the highest β-lactamase inhibitory activity and giving zones of inhibition in the hole-in-plate assay were combined, concentrated and then desalted on a Biorad Biogel P2 column. These fractions were shown to contain clavulanic acid by paper and thin layer chromatography.

EXAMPLE 19 ISOLATION OF SOLID CLAVULANIC ACID SODIUM SALT

A partially purified solid preparation of clavulanic acid (500 mg) prepared as in Example 17 was loaded onto a Whatman microgranular CC 31 cellulose column with 1 × 20 inches bed size. The chromatographic solvent was n-butanol/ethanol/water 4:1:5 v/v, top phase. The column was run at 4° C and 4 ml. fractions collected. Fractions were tested for the presence of clavulanic acid by spotting onto filter paper and spraying with the Ehrlich (pink spot) or triphenyltetrazolium (red spot) spray reagents. These spot tests were confirmed by β-lactamase inhibition assays at a 1/1250 dilution. Active fractions were combined and dried under vacuum on a rotary evaporator. The solid was dissolved in a small volume of distilled water and freeze dried. A white solid preparation of the sodium salt of clavulanic acid was obtained (40 mg) having an I₅₀ of 0.08 βg/ml in the β-lactamase inhibition assay.

EXAMPLE 20 ISOLATION OF SOLID CLAVULANIC ACID SODIUM SALT

Concentrated back extract (6 l) (from ultrafiltration in Example 16) containing 10 g of clavulanic acid as determined by the β-lactamase inhibition assay of Description 1. This was percolated at 1 l/hr onto a 2 × 24 inches column of Permutit Zerolit FF 1 P SRA 62 anion exchange resin in the chloride form. The column was then washed with 2 l of deionized water prior to elution with a sodium chloride gradient. The gradient was formed by a reservoir containing 4 l of 1.4 m NaCl feeding a stirred reservoir containing 4 l of 0.7 NaCl which in turn was connected to a stirred reservoir containing 4 l of deionized water which was connected via a pump to the column. The column was eluted at 2.5 ml/min and 25 ml fractions collected. Fractions were assayed by the β-lactamase inhibition assay.

Active fractions (nos. 140-230) were combined and vacuum evaporated to near dryness. Ethanol (500 mls) was then added and the solid filtered off after vigorous shaking. The ethanol extract was then vacuum evaporated to dryness on a rotary evaporator and redissolved in deionized water (40 mls). This was loaded onto a 4 × 24 inches column of Biorad Biogel P₂ and eluted with a 1% n-butanol solution. Fractions were collected (25 ml) and assayed for β-lactamase inhibitory activity at a 1/2500 final dilution. Tests for sodium chloride content on 1/25 dilutions of the fractions were made using silver nitrate solution. Those fractions containing clavulanic acid free of sodium chloride were combined, concentrated by evaporation of the solvent under reduced pressure to 20 mls and then freeze dried. This yielded 4.8 g of the sodium salt of clavulanic acid. (I₅₀ about 0.06 μg/ml)

EXAMPLE 21 PREPARATION OF AN ESTER OF CLAVULANIC ACID (METHYL ESTER) ##STR13##

19.8 mg. of the sodium salt of clavulanic acid was dissolved in 0.5 ml. dry dimethylformamide and treated with 0.25 ml. methyl iodide. After standing at room temperature for 1.5 hours under anhydrous conditions, the solvents were removed in vacuo. The residue was purified by P.L.C. on silica gel (Kieselgel 60F254 supplied by E. Merck, Darmstadt, Germany) eluting with ethyl acetate to give clavulanic acid methyl ester as a colourless oil (R_(f) 0.38; red color with triphenyltetrazolium chloride spray) which had the following properties:

Analysis: Found C 50.49 H 5.43 N 6.29

C₉ H₁₁ NO₅ Requires C 50.70 H 5.20 N 6.57

λ max (Methanol): no absorption >215 nm

ν max (Film): 3300 - 3600 (Broad), 1800, 1750, 1695 cm⁻¹

Approximate 1st order N.M.R. (CDCl₃): 2.49 (broad S, 1, exchanged with D₂ O), 3.05

(d, 1, J = 17.5 Hz), 3.54 (dd, 1, J = 17.5 Hz, J₂ = 2.5 Hz), 3.84 (S, 3)

4.24 (d, 2, J = 7 Hz), 4.93 (dt, 1, J = 7 Hz, J₂ = 1.5 Hz), 5.07 (d, 1, J = 1.5 Hz), 5.72 (d, 1, J = 2.5 Hz)

Molecular weight (mass spectrum): 213.0635. Calculated for C₉ H₁₁ NO₅ : 213.0637

Thin layer chromatography of the methyl ester showed a single zone in each of the following solvent systems; butanol/ethanol/water 4:1:5 v/v top phase R_(f) 0.75; isopropanol/water, 7:3 v/v R_(f) 0.95; ethylacetate/ethylalcohol 8:2 v/v R_(f) 0.87. The zones were detected by bioautography using Klebsiella aerogenes with added benzylpenicillin (synergism system).

EXAMPLE 22 PREPARATION OF AN ESTER OF CLAVULANIC ACID (p-nitrobenzyl ester) ##STR14## Treatment of the sodium salt of clavulanic acid with p-nitrobenzyl bromide in dry DMF gave, after P.L.C., a colorless oil which crystallized from chloroform - ether to give to p-nitrobenzyl ester of clavulanic acid as white feathery needles, m.p. 111 - 112° C, which on recrystallization had a mp of 117.5 - 118° C. EXAMPLE 23 PREPARATION OF AN ESTER OF CLAVULANIC ACID (BENZYL ESTER) ##STR15##

Impure 3-(β-hydroxyethylidine)-7-oxo-4-oxa-1-azabicyclo [3,2,0]heptane-2-carboxylic acid sodium salt (thought to be roughly 55 mg. of pure material) in dry dimethylformamide (0.64 ml.) was treated with benzyl bromide (0.18 ml.). The solution was kept at room temperature (approx. 17°-18° C) for 3 hours under anhydrous conditions. The reaction mixture was fractionated on silica gel, eluting with ethyl acetate, to give in substantially pure form the benzyl ester of 3-(β-hydroxyethylidine)-7-oxo-4-oxa-1-azabicyclo [3,2,0]heptane-2-carboxylic acid 63 mg.) as a colorless oil. i.r. (film) 1800, 1745, 1695 cm⁻¹ ; n.m.r. (CDCl₃), 2.25 (s,1, exchangeable with D₂ O), 3.05 (d,1,J = 17Hz), 3.51 (dd,1,J=17 Hz, J₂ =2.5 Hz), 4.24 (d,2,J=7.5Hz), 4.92 (dt, 1,J=7.5Hz, J₂ =1.5Hz), 5.15 (d,1,J=1.5Hz), 5.24 (s,2), 5.71. (d,1,J=2.5 Hz), 7.45 δ (s,5).

EXAMPLE 24 PREPARATION OF THE BENZYL ESTER OF CLAVULANIC ACID FROM CRUDE EXTRACTS OF THE FULTURE FILTRATE OF S. CLAVULIGERUS

Culture filtrate 20 l. obtained as described in Example 10 was vacuum evaporated using a climbing film evaporator to 5 l. The concentrate was then freeze-dried using an Edwards E.F.6 shelf freeze-drier manufactured by Edwards High Vacuum Ltd. The 300g. of solid so obtained contained 3 g. of sodium Clavulanic acid as determined by the enzyme inhibition assay. The solid was suspended in 900 ml. of dry dimethylformamide and 150 ml. of benzyl bromide was added. The mixture was stirred for 2 hours at room temperature and then diluted with 1 l. of ethyl acetate. The reaction mixture was filtered and the filtrate concentrated to as low a volume as was possible. The oily residue was extracted with a further 1 l. of ethyl acetate and the extract filtered. The filtrate was again concentrated and the resulting oily residue loaded onto a 3 × 14 inches silica gel column (Biogel Biosil A 100 mesh) in cyclohexane. The column was eluted with cyclohexane to remove benzyl bromide and the solvent was then changed to ethyl acetate and 20 ml. fractions collected. Fractions were tested for the presence of the benzyl ester of clavulanic acid by spotting onto glass backed silica gel t.l.c. plates (Merck precoated silica gel 60 F 254) and spraying with 2,3,5-triphenyl-tetrazolium chloride (TTC) spray reagent.

EXAMPLE 25 PREPARATION OF CLAVULANIC ACID BENZYL ESTER

Spray dried solid (3.3 kg) containing 69.4 g of clavulanic acid as determined by enzyme inhibition assay was obtained as described in Example 16. The solid was slurried in 5.5 l. of dimethylformamide and 500 mls. of benzyl bromide added. After stirring at room temperature for 2 hours, 12 l. of ethyl acetate were added and the solids removed by filtration. The filtrate was vacuum evaporated to an oily residue (212 g). The residue was loaded onto a column containing a 4 × 13 inches bed of silica gel (Hopkins & Williams MFC) in cyclohexane. The column was eluted with 12 l. of cyclohexane to remove excess benzyl bromide. The eluent was then changed to ethyl acetate and 500 ml. fractions collected. These were tested for benzyl clavulanate content by spotting onto silica gel t l c plates (Merck precoated silica gel 60 F 254) and spraying with 2,3,5 triphenyltetrazolium chloride (TTC) spray reagent. Fractions giving intense red spots were further examined by t l c on silica gel with chloroform/ethyl acetate 8:2 as the solvent and spraying the developed plates with T T C spray. Fractions 5-13 contained the bulk of the ester, and these were combined and vacuum concentrated to an oil (79.3 g). This preparation was then chromatographed on a 4 × 18 inches column of silica gel (Merck silica gel H type 60) with chloroform/ethyl acetate 8:2 as the solvent. Fractions Fractions giving intense red spots with this reagent were further examined by t.l.c. on silica gel plates using chloroform-ethyl acetate 8:2 as the solvent and spraying the developed plates with TTC spray. The benzyl ester of clavulanic acid runs at R_(f) 0.31 at 22° C. Fractions containing this ester were combined and concentrated to 15 ml. and this solution was further chromatographed on a 11/2 × 16 inches silica gel column (Merck silica gel H, type 60) with chloroform/ethyl acetate 8:2 as the solvent. 15 ml. fractions were collected and tested for the benzyl ester as described above. Those fractions containing the ester were concentrated to 8 ml. and finally purified by column chromatography on a 1 × 16 inches silica gel column (Merck silica gel H, type 60) with ethyl acetate cyclohexane 8:2 as the solvent. Selected fractions were combined and vacuum evaporated to give pure benzyl ester as an oil, 160 mg. were selected as described above and yielded on concentration 45.9g. of oil which was of 62% purity as adjudged by NMR spectroscopy

This product was finally chromatographed on a 23/4 × 18 inches column of Sephadex LH 20 (Pharmacia) in cyclohexane/chloroform 1:1. After selection of fractions and concentration a colourless oil (27.6 g) was obtained which proved to be 95% pure benzyl ester of clavulanic acid as determined by NMR spectroscopic examination.

EXAMPLE 26 PREPARATION OF CLAVULANIC ACID BENZYL ESTER

Culture filtrate (150 l.) pH 7.0 contained 16.2 g. of clavulanic acid (sodium salt) as determined by the enzyme inhibition assay. This filtrate was stirred with 5 kg. of Amberlyst A.26 anion exchange resin in the chloride form (Rohm & Haas Company: Philadelphia, USA) for 1 hour at room temperature. The resin was then filtered and the filtrate reassayed, showing that 6.4 g of clavulanic acid had been removed. The resin was washed with 20 l. of deionized water followed by 20 l. of acetone and 10 l. of dimethyl formamide (DMF). After refiltering the resin was suspended in 2.3.1. l. of DMF/0.2 M NaI. To this was added 200 mls. of benzyl bromide and the suspension stirred thoroughly. After standing at room temperature for 16 hours, ethyl acetate (2 l.) was added, and the resin then filtered, further washings (Ethyl acetate) of the resin were combined with the filtrate. The extract was then concentrated to a small volume and chromatographed on 3 × 18 inches silica gel column (Merck silica gel H type 60) with ethyl acetate/cyclohexane 8:2 as the solvent. Fractions containing benzyl clavulanate were selected by spotting onto silica gel t l c plates and spraying with TTC reagent as described previously (Example 24). Those selected were concentrated to 20 mls and then chromatographed on a 11/2 × 18 inches silica gel column (Merck silica gel H type 60) with chloroform/ethyl acetate 8:2 as the solvent. Selected fractions were combined and evaporated to a colourless oil (440 mgs) which was 90% benzyl clavulanate as determined by NMR spectroscopy.

EXAMPLE 27 PREPARATION OF THE BENZYL ESTER OF CLAVULANIC ACID FROM CRUDE EXTRACTS OF THE CULTURE FILTRATE OF S. CLAVULIGERUS

An aliquot of aqueous back extract of the butanol extract of culture filtrate obtained as described in Example 14 was freeze-dried using an Edwards chamber drier. A 24 g. portion of the solid obtained contained 0.96 g. of sodium clavulanic acid as determined by the enzyme inhibition assay. This solid was suspended in 75 ml. of dry dimethylformamide and 75 ml. of benzyl bromide was added. The mixture was stirred for 2 hours at room temperature. The suspension was then diluted with 500 ml. of ethyl acetate and the mixture filtered. The filtrate was concentrated to an oily residue on a vacuum rotary evaporator. This residue was loaded onto a 2 × 14 inches silica gel column (Biogel Biosil A.100 mesh) in cyclohexane. Benzyl bromide was eluted from the column and then the solvent was changed to ethyl acetate and 10 ml. fractions was collected. Fractions containing the benzyl ester of clavulanic acid were selected as in Example 24. Further purification was also achieved as described in Example 24 by column chromatography. This process yielded 220 mg. of pure benzyl ester.

EXAMPLE 28 PREPARATION OF CLAVULANIC ACID SODIUM SALT ##STR16##

Substantially pure benzyl clavulanate (281 mg) in ethanol (25 ml.) containing sodium hydrogen carbonate (82 mg.) was hydrogenated over 10% Pd/C (90 mg.) for 25 minutes at room temperature and atmospheric pressure. The catalyst was filtered off, washed with water and ethanol, and the combined filtrates evaporated under reduced pressure at room temperature. The residual semi-solid was triturated with acetone, filtered and washed with ether to yield sodium clavulanate (135 mg.)

EXAMPLE 29 HYDROLYSIS OF CLAVULANIC ACID METHYL ESTER TO CLAVULANIC ACID

2.17 mg. of clavulanic acid ester was dissolved in 0.1 ml. methanol and treated with 0.208 ml. sodium hydroxide solution (0.0482N). After 1 hour at room temperature, the reaction mixture contained several products. T.L.C. analysis indicated that one of the major components had an R_(f) identical to that of the sodium salt of clavulanic acid; color reactions and biological assay were consistent with this component being the sodium salt of clavulanic acid.

Slow conversion of the ester to clavulanic acid was seen when 1 mg/ml. of the compound was incubated at 37° C in 0.05M phosphate buffer at pH 7. The reaction was followed by paper chromatography (bioautographic system). Using the butanol/ethanol/water system to follow the reaction over a period of 2 hours the zone of the methyl ester at R_(f) 0.79 decreased in size as the zone of clavulanic acid at R_(f) 0.12 increased.

EXAMPLE 30 ANTIBACTERIAL SPECTRUM OF CLAVULANIC ACID

The antibacterial activity of clavulanic acid sodium salt against a range of bacteria was determined using the microtitre method. Serial dilutions of clavulanic acid sodium salt in Oxoid sensitivity test broth contained in a microtitre plastic tray were inoculated with an overnight broth culture of each organism so that the final dilution of the inoculum was 0.5 × 10⁻⁴. The cultures were incubated overnight and the points of bacterial growth recorded next morning by observing the turbidity of the culture. The results, expressed as approximate MIC values (minimum inhibitory concentration μg/ml.) are recorded in Table 3 which shows that the compound has a broad spectrum of antibacterial activity.

                  TABLE 3                                                          ______________________________________                                         ANTIBACTERIAL SPECTRUM OF CLAVULANIC ACID                                      SODIUM SALT                                                                    ______________________________________                                                              Minimum Inhibitory                                          Bacterial Strain   Concentration μg/ml.                                   ______________________________________                                         Staphylococcus aureus (Oxford H)                                                                    7.5                                                       Staphylococcus aureus (Russell)                                                                     7.5                                                       Bacillus subtilis    62                                                        Streptococcus faecalis                                                                              >500                                                      Streptococcus pyogenes CN 10                                                                        125                                                       Escherichia coli NCTC 10418                                                                         31                                                        Klebsiella aerogenes 31 - 62                                                   Klebsiella oxytocum  62                                                        Enterobacter aerogenes T 624                                                                        31                                                        Enterobacter cloacae 62                                                        Acinetobacter anitratus                                                                             125                                                       Providentia stuartii 125                                                       Serratia marcescens  125                                                       Proteus mirabilis 0977                                                                              62                                                        Proteus vulgaris W090                                                                               31                                                        Salmonella typhimurium                                                                              31                                                        Shigella sonnei      62                                                        Pseudomonas aeruginosa A                                                                            500                                                       ______________________________________                                    

EXAMPLE 31 EXAMPLES OF β-LACTAMASE INHIBITION BY CLAVULANIC ACID SODIUM SALT

Clavulanic acid progressively and irreversibly inhibits the β-lactamase of Escherichia coli. The method of Description 1 shows that the other β-lactamases shown in Table 4 are also inhibited by clavulanic acid.

                  TABLE 4                                                          ______________________________________                                         INHIBITION OF β-LACTAMASES BY CLAVULANIC ACID                             ______________________________________                                                           Approximate                                                                    I.sub.50 Value Relative to                                   Source of β-lactamase                                                                       Escherichia coli JT 4 = 1                                    ______________________________________                                         Staphylococcus aureus                                                                            1.0                                                          (Russell)                                                                      Escherichic coli JT4                                                                             1.0                                                          Escherichia coli B11                                                                             2.0                                                          Klebsiella aerogenes A                                                                           0.6                                                          Pseudomonas aeruginosa 1822                                                    (R factor)        5.0                                                          Pseudomonas dalgleish                                                                            0.1                                                          ______________________________________                                    

With pencillin G as substrate the I₅₀ of clavulanic acid sodium salt against the β-lactamase of Staph. aureus (Russell) is approximately 0.06 μg/ml.

EXAMPLE 32 EXAMPLES OF ACTIVITY OF CLAVULANIC ACID METHYL ESTER

Tests for antibacterial activity in both showed clavulanic acid methyl ester to have broad spectrum activity but of a lower order than shown by clavulanic acid. It was not clear whether this activity was the activity of the compound itself or of clavulanic acid liberated by slow aqueous hydrolysis of the ester. Clavulanic acid methyl ester showed marked antibacterial synergism in combination with ampicillin or cephaloridine against bacteria resistant to these antibiotics. Thus, the minimum inhibitory concentration (M.I.C.) for ampicillin against Staphylococcus aureus (Russell) was reduced from 500 μg/ml. to <0.4 in the presence of 1.0 μg/ml. clavulanic acid methyl ester. The M.I.C. for cephaloridine was reduced from 1.5 μg/ml. to <0.03 μg/ml. in the presence of 1 μg=/ml. of clavulanic acid methyl ester. The M.I.C. for ampicillin against Proteus mirabilis C889 was reduced from 500 μg/ml. to 31 μg/ml. in the presence of 5 μg/ml. clavulanic acid methyl ester.

EXAMPLE 33 PREPARATION PIVALOYLOXYMETHYL CLAVULANATE

To a stirred solution of bromomethyl pivalate (C.37g) in dry dimethylformamide (5 ml) was added sodium clavulanate (0.49g). After 2 hrs. at room temperature the reaction mixture was treated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (20 ml). The mixture separated into two layers and the non-aqueous layer was separated, washed with water (20 ml) and dried over sodium sulphate. The dried solution was evaporated to leave the required product as a pale yellow oil. (500 mg). N.m.r. (CDCl₃), 1.26 (s,9), 3.13 (d,1,J=17 Hz), 3.62 (dd, 1,J,=17Hz, J₁ =2.5Hz, 4.3(d,2,J=7.5Hz), 5.0 (dt, 1,J=7.5Hz, J₂ =1.5Hz), 5.16(d,1,J=1.5Hz), 5.79(d,1,J=2.5Hz), 5.92ν(s,2); i.r.(liquid film), υ β-lactam C.O 1800 cm⁻¹, ester C=O 1760 cm.sup. -1.

EXAMPLE 34 PREPARATION OF CLAVULANIC ACID PHTHALIDE ESTERS

To a stirred solution of 3-bromophthalide (0.43g) in dry dimethylformamide (5 ml) was added sodium clavulanate (0.5g) and the solution was left at room temperature for 2 hours. The solution was treated with ethyl acetate (20 ml), cyclohexane (10 ml) and water (30 ml) and shaken thoroughly. The non-aqueous layer was washed with water (20 ml), dried (Na₂ SO₄) and evaporated to yield a pale yellow gum. The two diastereomeric esters were separated using high pressure liquid chromatography on a 40 cm × 10 mm column of silica gel (Merckosorb SI 60, 5 μ) eluting with ethyl acetate at a flow rate of 3 ml/min.

The first phthalide ester (retention time 7.15 min) crystallized from ethyl acetate as needles, mp 102° , and had the following i.r. (Nujol mull) ν β-lactam C=O 1790 cm⁻¹ ester C=O 1755 cm⁻¹ n.m.r. (CD₃ COCD₃): 3.14 (d,1,J=17.Hz) 3.76 (dd,1,J,=17.5Hz, J₂ =2.5Hz), 4.25(d,2,J=7.5Hz), 5.0 (dt,1,J₁ =7.5Hz, J₂ =1.5Hz), 5.4 (s,1,J=1.5Hz), 5.82 (d,1,J=2.5Hz), 7.7 (s,1), 8.06δ(m,4); M.wt (mass spectrometry: 331.0696 corresponds to C₁₆ H₁₃ NO₇ (calc. 331.0692). The second diasterioisomer (retention time 8.85 min) had the following i.r. (CH₂ Cl₂ solution) ν β-lactam C=O 1800 cm⁻¹, ester C=O 1780 cm⁻¹ ; nmr (CDCl₃) 2.42 (broad S,1, exchangeable with D₂ O), 3.12 (d,1, J=18 Hz), 3.60 (dd,1,J₁ =18 Hz, J₂ =2.5Hz), 4.30 (d,2,J=7.5Hz), 5.0 (dt,1,J₁ =7.5Hz, J₂ =1.5Hz), 5.12 (d,1,J=1.5Hz), 5.76 (d,1,J=2.5 Hz), 7.52 (S,1), 7.85δ(m,4). ##STR17##

EXAMPLE 35 PREPARATION OF NONYL CLAVULANATE

Sodium clavulanate (44 mg) in dry dimethylformamide (2 ml) was treated with nonyl iodide (76 mg) and left at room temperature for 2 hours. The solution was evaporated and the residue fractionated on silica gel, eluting with ethyl acetate-hexane (2:1 to give the product as an oil; i.r. (film) 1800, 1745, 1690 cm⁻¹. M.wt. (mass spectrometry) = 325.1890 which corresponds to C₁₇ H₂₇ NO₅. (calc. 325.1889).

EXAMPLE 36 PREPARATION OF CLAVULANIC ACID

Benzyl clavulanate (100 mgs) in ethanol (5 ml) was hydrogenated over 10% Pd/C (30 mgs) for 45 minutes at ambient temperature and atmospheric pressure. The catalyst was filtered, washed with ethanol and the combined filtrates were evaporated in vacuo to give clavulanic acid as an unstable, viscous oil (58 mgs). N.m.r. (C₅ D₅ N): 3.05(d,1,J=18Hz), 3.60(dd,1,J₁ =18Hz, J₂ =2.5Hz), 4.75(d,2,J=7.5Hz), 5.58(t,1,J=7.5Hz), 5.66 (S,1), 6.0δ(d,1,J=18Hz).

EXAMPLE 37 PREPARATION OF METHYL CLAVULANATE

Clavulanic acid (130 mgs) in ethanol (10 ml) was treated with excess diazomethane in ether. After 2 minutes at room temperature the reaction was shown (t 1 c) to be complete. The solution was evaporated in vacuo and the residue purified by chromatography on silica gel, eluting with ethyl acetate. The fractions containing methyl clavulanate were combined and evaporated to give a clear oil (104 mgs).

EXAMPLE 38 PREPARATION OF METHYL CLAVULANATE

Clavulanic acid (200 mgs) in acetonitrile (5 ml) was cooled and stirred at 0°. Methanol (0.5 ml) and then dicyclohexyldicarbodiimide (206 mg.) were added and the reaction mixture was stirred at room temperature overnight. The suspension was filtered and the filtrate evaporated in vacuo to give crude methyl clavulanate. The crude product was purified by chromatography on silica gel, eluting with ethyl acetate, to give a clear oil (140 mg).

EXAMPLE 39 PREPARATION OF PHENYL CLAVULANATE

Clavulanic acid (100 mg) in acetonitrile (5 ml) was cooled and stirred at 0°. To the solution was added phenol (0.94g) and dicyclohexyldicarbodiimide(100 mg) and the reaction mixture was stirred at room temperature overnight. The suspension was filtered and the filtrate evaporated. The residue was fractionated on silica gel, eluting with ethyl acetate-hexane (1:1) to give phenyl clavulanate (70 mg). I.r (film) 1800, 1770, 1690 cm⁻¹. N.m.r. (CDCl₃)2.18 (broad s,1), 3.06 (dd, 1,J=17Hz,J₂ =0.9Hz), 3.54 (dd,1,J₁ =17Hz,J₂ =2.6Hz), 4.29 (d,2,J=7.5Hz), 5.1(dt,1,J₁ =7.5Hz,J₂ =1.5Hz) 5.29 (d,1,J=1.5Hz),5.76(dd,1,J₁ =2.6Hz,J₂ =0.9Hz), 7.35δ(m,5). M.wt. (mass spectrometry) = 275.0777 which corresponds to C₁₄ H₁₃ NO₅ (calc. 275.0794).

EXAMPLE 40 PREPARATION OF 2,2,2-trichloroethyl clavulanate

Sodium clavulanate (221 mgs) was suspended in dry tetrohydrofuran (5 mls) and stirred at 0°. Trichloroethylchloroformate (211 mg) in dry tetrohydrofuran (1 ml) was added to the above suspension over 20 minutes. The mixture was allowed to reach room temperature and stirred overnight. The suspension was filtered and the filtrate evaporated in vacuo. The residue was chromatographed on silica gel eluting with ethyl acetate - hexane (2:1) to give the required product as an oil. i.r (film) 1800, 1760, 1690 cm⁻¹. n.m.r. (CDCl₃) 1.56 (broad S,1), 3.07 (dd,1,J₁ =17.5Hz,J₂ =0.7Hz), 3.56 (dd,1,J₁ =17.5Hz, J₂ =2.5Hz), 4.24 (d,2,J=7.5Hz), 4.69 (d,1,J=12Hz), 4.92 (d,1,J=12Hz), 5.02 (dt,1,J₁ =7.5Hz, J₂ =1.3Hz), 5.19(d,1,J=1.3Hz), 5.73 δ (dd,1,J₁ =2.5Hz, J₂ =0.7Hz). M.wt. (mass spectrometry) = 328.9621 which corresponds to C₁₀ H₁₀ NO₅ Cl₃ (calculated 328.9625). ##STR18##

EXAMPLE 41 PREPARATION OF SODIUM CLAVULANATE

Benzyl clavulanate (840 mgs) in ethanol (30 ml) and water (5 ml ) was hydrogenated over 10% Pd/C (267 mgs) and sodium bicarbonate (244 mgs) for 25 minutes at room temperature and atmospheric pressure. The catalyst was filtered, washed with water and ethanol and the combined filtrates were evaporated in vacuo. The product crystallized from a water-acetone mixture as microneedles (565 mgs). Recrystallization from water-acetone gave needles which, after drying over P₂ O₅ in vacuo for 24 hours gave the following analysis: C41.01, 40.86; H 3.77, 3.64; N 5.68, 5.51; i.r.(KBr disc) 1785, 1700, 1620 cm⁻¹ ; Nmr (D₂ O) 3.06 (d,1,J=18.5Hz), 3.57 (dd,1,J₁ =18.5Hz, J₂ =2.5Hz), 4.15 (d,2,J=8Hz), 5.3 (HOD), 4.9(m), 5.71 (d,1,J=2.5Hz)

EXAMPLE 42 ANTIBACTERIAL SYNERGISM BETWEEN AMPLICILLIN AND CLAVULANIC ACID SODIUM SALT

The minimum inhibitory concentration (M.I.C. values) of ampicillin, clavulanic acid sodium salt and ampicillin in the presence of 1 μg/ml. clavulanic acid sodium salt were determined for a range of β-lactamase producing bacteria. The organisms were inoculated into Oxoid sensitivity test broth located in small wells in a plastic tray and containing separate concentration gradients of ampicillin, clavulanic acid sodium salt or ampicillin plus 1 μg/ml. clavulanic acid sodium salt (microtitre method). The final dilution of the inoculum was 0.5 × 10⁻². The tray was incubated at 37° C overnight and a record made next morning of the end points of bacterial growth. The M.I.C. values in μg/ml. are recorded in Table 5 which reveals that the synergist at the low concentration of 1 μg/ml. markedly enhances the antibacterial activity and ampicillin against certain Gram negative and Gram positive bacteria. The mechanism of this synergism is likely to involve inhibition of ampicillin destroying β-lactamase enzymes but the existence of other mechanisms cannot be excluded.

Similar results to those shown in Table 5 were obtained when ampicillin was replaced by amoxycillin or by the phthalidyl ester of ampicillin.

                  TABLE 5                                                          ______________________________________                                         ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND                                 CLAVULANIC ACID SODIUM SALT                                                    ______________________________________                                                   Minimum Inhibitory Concentrations μg/ml                                                           Ampicillin in                                              Clavulanic          presence of                                                acid                lμg/ml. clavu-                                          sodium              lanic acid                                     Bacterial strain                                                                           salt      Ampicillin                                                                               sodium salt                                    ______________________________________                                         Escherichia coli                                                               NCTC 10481  31        1.8       <0.4                                           Escherichia coli                                                               B 11        62        >500      125                                            Klebsiella                                                                     aerogenes A 31        125       <0.4                                           Klebsiella sp 62                                                                           31        125       <0.4                                           Enterobacter                                                                   cloacae     62        250       62                                             Serratia                                                                       marcescens  125       >500      62                                             Staphylococcus                                                                 aureus(Russell)                                                                            15        500       <0.4                                           Staphylococcus                                                                 aureus      62        250       7.5                                            ______________________________________                                    

EXAMPLE 43 ANTIBACTERIAL SYNERGYSM BETWEEN CEPHALORIDINE AND CLAVULANIC ACID SODIUM SALT

The minimum inhibitory concentrations of cephaloridine, clavulanic acid sodium salt and cephaloridine in the presence of 5 μg/ml clavulanic acid sodium salt were determined by the method described in Example 42. The results in Table 6 show that synergism can be obtained between clavulanic acid sodium salt and cephaloridine particularly for the β-lactamase producing strain of Staphylococcus aureus (Russell).

                  TABLE 6                                                          ______________________________________                                         ANTIBACTERIAL SYNERGISM BETWEEN CEPHALORIDINE                                  AND CLAVULANIC ACID SODIUM SALT                                                ______________________________________                                                   Minimum Inhibitory Concentrations μg/ml.                                      Clavulanic         Cephaloridine in                                            acid               presence of                                     Bacterial   sodium    Cephal-  5μg/ml clavulanic                            Strain      salt      ridine   acid sodium salt                                ______________________________________                                         Proteus                                                                        mirabilis                                                                      899         <500*     62       7.5                                             Staphylococcus                                                                 aureus(Russell)                                                                            15        3.1      >0.03.sup.+                                     Staphylococcus                                                                 aureus      62        15       3.7                                             ______________________________________                                          *Tailing Point                                                                 .sup.+ Same value obtained when synergist added at 1 μg/ml. instead of      5 μg/ml.                                                              

EXAMPLE 44 ANTIBACTERIAL SYNERGISM BETWEEN CLAVULANIC ACID SODIUM SALT AND VARIOUS PENICILLINS

The results presented in Table 7 were obtained by the method described in Example 42.

                  TABLE 7                                                          ______________________________________                                         ANTIBACTERIAL                                                                  SYNERGISM BETWEEN CLAVULANIC ACID                                              SODIUM SALT AND VARIOUS PENICILLINS AGAINST                                    STRAINS OF KLEBSIELLA AEROGENES                                                ______________________________________                                         Amoxycillin    Carbenicillin*                                                                              Benzylpenicillin                                                 +5μg/ml.    +5μg/ml.  +5μg/ml.                          Strain                                                                               Alone   synergist                                                                               Alone synergist                                                                             Alone synergist                            ______________________________________                                         A     500      0.97    500   7.8    250    7.8                                 E 70  500     3.9      500   15     500   15.6                                 62    250     15.6     125   7.8    250   15.6                                 ______________________________________                                          *Similar results observed when carbenicillin replaced by carbenicillin         phenyl α-ester or ticacillin.                                      

EXAMPLE 45 ANTIBACTERIAL SYNERGISM BETWEEN AMPICILLIN AND ESTERS OF CLAVULANIC ACID

The results presented in Table 8 were obtained by the method described in Example 42

                  TABLE 8                                                          ______________________________________                                         ANTIBACTERIAL                                                                  SYNERGISM BETWEEN AMPICILLIN AND                                               ESTERS OF CLAVULANIC ACID AGAINST STRAINS OF                                   KLEBSIELIA AEROGENES                                                           ______________________________________                                                             Ampicillin + Ampicillin +                                                      5 μg/ml of                                                                               5 μg/ml of                                                     Methyl Ester Benzyl Ester                                                      of           of                                                    Ampicillin  clavulanic   clavulanic                                    Strain  Alone       acid         acid                                          ______________________________________                                         A       500         1.9          1.9                                           E 70    500         3.9          3.9                                           62      500         3.9          3.9                                           ______________________________________                                    

Neither clavulanic acid methyl ester nor clavulanic acid benzyl ester inhibited the growth of the test organisms at a concentration of 100 μg/ml.

EXAMPLE 46 ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID ESTER

The method of Example 30 but using a dilution of 1/100 of overnight broth, the MIC values in Table 9 were obtained for certain esters of clavulanic acid against a number of organisms:

                                      TABLE 9                                      __________________________________________________________________________     ANTIBACTERIAL ACTIVITY OF CLAVULANIC ACID ESTERS                                                               MIC*                                                                           of                                                     MIC of Ester of Clavulanic Acid                                                                        clavulanic                                             Benzyl                                                                              Nonyl                                                                              Pivaloyloxy-                                                                           Phthalidyl                                                                            acid sodium                                    Organism                                                                               ester                                                                               ester                                                                              methyl ester                                                                           ester  salt                                           __________________________________________________________________________     Bacillus                                                                       subtilis A                                                                             250  31  62      125    62                                             Staph.aureus                                                                   Oxford   62  31  31       31    15                                             Staph.aureus                                                                   Russell 125  31  62       15    15                                             Escherichia                                                                    coli 10418                                                                             125   250                                                                               125     125    125                                            __________________________________________________________________________      *The MIC of clavulanic acid sodium salt is included for comparison: the        high MIC values (if compared to those of Example 30) are due to the heavy      inocula used.                                                            

EXAMPLE 47 EXTRACTION OF CLAVULANIC ACID USING LIQUID ION EXCHANGE RESIN

Culture filtrate (200 ml, obtained in a similar manner to Example 3 but using a medium containing 0.1% v/v KH₂ PO₄ instead of 0.01% FeSO₄.7H₂ O) was extracted with Amberlite* LA2⁺ (Cl⁻ form, 15% v/v in methylisobutyl ketone, 66 ml) for 30 minutes at 5° C.

The phases were separated by centrifugation (1660 g, 20 minutes). The solvent phase (60 ml) was recovered by pipette and divided into four equal portions. Each portion was extracted by stirring at 5° C for 20 minutes with one-fourth volume (3.75 ml) aqueous extractant as indicated in the table below. The resulting mixture was centrifuged (1660 g, 15 minutes). 3.6 ml. aqueous phase was recovered from each extraction.

    ______________________________________                                                             Clavulanic acid                                                        Volume  concentration Clavulanic                                   Sample      (ml)    (μg ml.sup.-1)                                                                            acid (mg)                                    ______________________________________                                         clarified brew                                                                             200     128           25.4                                         extracted brew                                                                             200      15           3.0                                          M NaCl extract                                                                             3.6     305           1.1                                          2M NaCl extract                                                                            3.6     598           2.5                                          M NaNO.sub.3 extract                                                                       3.6     638           2.3                                          2M NaNO.sub.3 extract                                                                      3.6     758            2.73                                        ______________________________________                                    

The result obtained with 2M NaNO₃ represents a recovery of 43% from clarified brew.

EXAMPLE 48 EXTRACTION OF CLAVULANIC ACID USING LIQUID ION EXCHANGE RESIN

Clarified brew (47 liters, obtained as in Example 12) was extracted with Amberlite LA2 (acetate form, 15% v/v in methylisobutyl ketone, 12.5 liters) by stirring for 1 hour at 17° C. After adding octan-1-ol (500 ml) the phases were separated in a continuous flow centrifuge yielding 9.2 liters solvent phase, which was then stirred at 5° C for 11/2 hours with molar sodium nitrate (2.3 liters). The mixture was separated by continuous flow centrifugation yielding 2.4 liters aqueous phase (including water used for displacement purposes). Aqueous phase pH (initially 8.0) was adjusted to 7.0 with concentrated hydrochloric acid.

    ______________________________________                                                             clavulanic acid                                                                              clavulanic                                              Volume   concentration acid                                         Sample     (1)      (μg ml.sup.-1)                                                                            (mg)                                         ______________________________________                                         clarified brew                                                                            47       146           6862                                         extracted brew                                                                            47        19            893                                         M NaNO.sub.3 extract                                                                      2.4      1638          3931                                         ______________________________________                                    

Extraction efficiency from clarified brew to sodium nitrate extract is 57% ##SPC1##10/132

Clavulanic acid is thus a β-lactamase inhibitor and an antibacterial agent.

We have now discovered a group of derivatives of clavulanic acid which have antibacterial activity and some of which are useful in the preparation of deoxyclavulanic acid which is the compound of the formula (II): ##STR19##

A subgroup of compounds of the formula (II) include those of the formula: ##STR20##

Such compounds are particularly useful when formulated into pharmaceutical veterinary form for the treatment of diseases of the urinary tract, respiratory system and soft tissue as well as diseases such as otitis media, mastitus and the like. These compounds may be present in such compositions as a sole therapeutic agent or they may be present together with other therapeutic agents such as a β-lactam antibiotic.

Suitable β-lactam antibiotics for inclusion in such compositions include not only those known to be susceptible to β-lactamases but also those which have a good degree of intrinsic resistance to β-lactamases. Thus, suitable β-lactam antibiotics for inclusion in the composition of this invention include benzylpenicillin, phenoxymethylpenicillin, carbenicillin, methicillin, propicillin, hetacillin, ampicillin, 6-aminopenicillanic acid, 7-aminocephalosporanic acid, amoxycillin, ticarcillin, cephaloridine, cephalothin, cephalexin, cefoxitin, cephaloglycine, 7-aminodesacetoxycephalorporanic acid, cephamandole and in-vivo hydrolysable esters of such compounds such as the phenyl, tolyl and indanyl esters of carbenicillin and ticarcillin, the acetoxymethyl ester of benzylpenicillin and the pivaloyloxymethyl and phthalyl ester of ampicillin, amoxycillin, cephaloglycin cephalexin and the like.

When present in a pharmaceutical composition together with a β-lactam antibiotic, the ratio of the compound of formula (I) present to β-lactam antibiotic present may be from, for example, 20:1 to 1:5, such as 10:1 to 1:3 and advantageously may be 3:1 to 1:2.

The total quantity of antibacterial agents present in any unit dosage form will normally be between 50 and 1500 mg. and will usually be between 100 and 1000 mg. However, injectable or infusable compositions may contain greater quantities if desired, for example, 4g. or more of active material. Normally, between 50 and 6000 mg. of the compositions of this invention will be administered each day of treatment but more usually between 500 and 3000 mg. of the composition of the invention will be administered per day. However, for the treatment of severe systemic infections or infections of particularly intransigent organisms, such as Pseudomonas aeroginosa higher doses may be used in accordance with clinical practice.

Synergistic combinations contain deoxyclavulanic acid or a salt or ester thereof and ampicillin, amoxycillin or a pro-drug for ampicillin of amoxycillin. Such compositions are preferably adapted for administration to humans and contain 50 - 500 mg. of a salt or in-vivo hydrolysable ester of a compound of the formula (I) and 200 -1000 mg. of the penicillin. Such compositions may be used in treating infections of the urinary tract and the respiratory tract and are particularly useful in treating infections due to Klebsiella aeroginosa.

Synergistic combinations may also contain deoxyclavulanic acid or a salt or ester thereof and carbenicillin or ticarcillin or a pro-drug for carbenicillin or ticarcillin. Such compositions are preferably adapted for administration to humans and contain 50 - 1500 mg. of a salt or in-vivo hydrolysable ester of a compound of the formula (I) and 200 - 1500 mg. of the penicillin. Such compositions may be used in treating infections of the urinary tract and are particularly useful in treating infections due to gram-negative organisms such as Pseudomonas aeroginosa.

Deoxyclavulanic acid may be produced by the hydrogenation of a compound of the formula ##STR21##

Normally, such a reaction takes place in the presence of a transition metal catalyst such as palladium or the like. A particularly suitable catalyst is palladium on charcoal, for example, 10% palladium charcoal. The catalyst employed is preferably in highly active form, for example, the sort obtained by using a fresh batch of catalyst.

The process of this invention normally takes place in solution in an inert solvent at a non-extreme temperature; for example, the reaction may take place in a lower alkanol at a temperature of -10° C to +50 ° C, more usually from 0° C to 25° C, for example, from 5° C to 20° C. A high, medium or low pressure of hydrogen may be used in this reaction. Generally, it is preferred to use an atmospheric or slightly super atmospheric pressure of hydrogen.

A preferred form of the process comprises the hydrogenation in the presence of a palladium catalyst. The following example illustrates this process:

EXAMPLE 49 ##STR22##

Benzyl clavulanate (220 mg.) in ethanol (20 ml.) was hydrogenated over 10% Pd/C (70 mg.) and sodium hydrogen carbonate (60 mg.) for 60 minutes. The catalyst was filtered, washed with water and then ethanol and the combined filtrates were evaporated. This material was chromatographed on a silica gel column with n-butanol/ethanol/water; 4:1:1 3/4 and the fastest moving component was collected. The solvents were removed under low pressure to yield the sodium salt of de-oxyclavulanic acid (48 mg.). Esterification of the sodium salt in dimethylformamide with p-bormobenzylbromide yielded a liquid p-bromobenzyl ester. The palladium catalyst used in this reaction was supplied by Englehard, Chemcial Division, Valley Road, Cinderford, Gloucestershire, U.K.

There are also two isomeric acids of deoxyclavulanic acid of the formulas ##STR23## which are useful for the same purposes as deoxyclavulanic acid.

Accordingly the present invention provides compounds of the formula (III): ##STR24## wherein R is an optionally substituted C₁₋₄ alkyl, phenyl or tolyl group or a group O⁻ H⁺ M wherein M is a tertiary amine and CO₂ R¹ is an ester group.

Suitable substituents for R are chlorine, bromine or fluorine atoms or methoxy, ethoxy or acetoxy groups.

Preferred values for R include methyl, ethyl, phenyl, tolyl, chlorophenyl and methoxyphenyl groups.

Other perferred values for R include O⁻ H⁺ M groups wherein M is a pharamaceutically acceptable tertiary amine group that can complex with sulphur trioxide and contains up to 13 carbon atoms. Suitable such tertiary amine groups include trimethylamine, dimethylamine, pyridine, N-methylpiperidine and N-methylmorpholine groups.

Suitably CO₂ R¹ is an ester group as described above with reference to clavulanic acid. Preferably R¹ is a benzyl group.

One preferred group of compounds of the present invention are those of the formula (IV): ##STR25## wherein R² is a methyl, phenyl or tolyl group.

A second preferred group of compounds of the present invention are those of the formula (V): ##STR26## wherein M¹ is a trimethylamine, dimethylalanine, pyridine, N-methylpyridine or N-methylmorpholine group.

Preferably M¹ is a trimethylamine group.

The compounds of the formula (III) have some antibacterial activity against certain strains of β-lactamase producing bacteria. Thus in one of its aspects the present invention provides pharmaceutical compositions comprising a compound of the formula (III) together with a pharmaceutically acceptable carrier.

The compounds of the formula (III) are also useful intermediates in the preparation of deoxyclavulanic acid, the compound of the formula (II), and its derivatives.

Deoxyclavulanic acid may be prepared by the hydrogenation of a compound of the formula (III) in a solvent in the presence of a transition metal catalyst.

The benzyl clavulanyl sulphate trimethylammonium salt is converted to deoxyclavulanic acid by hydrogenation for 15 minutes at ambient temperature and pressure in a 50% ethanol/tetrahydrofuran solution in the presence of 10% palladium on charcoal.

The compounds of the formula (III) may be prepared by the reaction of a compound of the formula (VI): ##STR27## with a sulphonic acid HO.SO₂ R or a reactive sulphonylating derivative thereof or with sulphur trioxide in the presence of a tertiary amine.

Compounds of formula (VI) are available by using the methods described above with reference to clavulanic acid.

Suitable reactive sulphonylating derivatives of the acid HO.SO₂ R include the acid halides and pseudohalides such as R.SO₂ Cl, R.SO₂ Br, R.SO₂ I, R.SO₂ F, R.SO₂ N₃ and the like and acid anhydrides such as R.SO₂.O.SO₂ R and derivatives formed from the acid and a dehydrating agent such as a carbodiimide and the like.

Normally, the reaction is carried out in the presence of at least one mole of base; which is preferably a tertiary amine.

The reaction may take place at low, ambient or elevated temperatures, e.g. -20°-120° C, but in general moderate temperatures in the order of 0°-25° C are used.

In general the reaction is carried out in aprotic solvent systems such as dimethylformamide, dimethylsulphoxide, dioxan, tetrahydrofuran or the like.

The reaction of the compound of formula (VI) with sulphur trioxide takes place in the presence of a conventional tertiary amine.

Normally, a sulphur trioxide/amine complex is used.

The reaction takes place under similar physical conditions as outlined above for reaction with HO.SO₂ R or its derivatives.

The following Example illustrates the invention:

EXAMPLE 50 Preparation of Benzyl Clavulanate -- O -- sulphonate trimethylamine salt

Benzyl clavulanate (VII) (57.8 mg) in dry dimethylformamide (0.8 ml) was treated with the trimethylamine -- sulphur trioxide complex (55.6 mg) and left at ambient temperature for 18 hours. The solvent was removed in vacuo and the residue extracted with chloroform. The chloroform solution was evaporated and the residue was extracted several times with ether to remove benzyl clavulanate. The ether insoluble oil was shown to be (VIII) the trimethylamine salt of benzyl clavulanate -- o -- sulphonate, which was produced in 60% yield. (In the n.m.r. the --CH₂ OSO₃ ⁻ proton appeared at a doublet at about 4.71δ as compared to about 4.24δ in the starting material).

The preceeding reaction may be summarised thus: ##STR28##

The I₅₀ in μg/ml of this compound against several β-lactamases was determined using the method described in Belgian Pat. No. 827926 and the results are as follows:

    ______________________________________                                         Lactamase              I.sub.50 (μg/ml)                                     ______________________________________                                         Escherischia coli JT4  3.4                                                     Citrobacter freundii Mantio                                                                           2.2                                                     Pseudomonas aeruginosa 1.1                                                     Pseudomonas Dalgleish  4.2                                                     ______________________________________                                     

What we claim is:
 1. A compound of the formula (V): ##STR29## wherein M¹ is trimethylamine, dimethylalanine, pyridine, N-methyl-pyridine or N-methylmorpholino.
 2. A compound according to claim 1 wherein M¹ is trimethyl. 